PRETARGETING METHODS AND COMPOUNDS

Abstract: Methods, compounds, compositions and kits that relate to pretargeted delivery of diagnostic and therapeutic agents are disclosed. In particular, methods for radiometal labeling of biotin, as well as related compounds, are described. Articles of manufacture useful in pretargeting methods are also discussed.

Description: PRETARGETING METHODS AND COMPOUNDS Technical Field The present invention relates to methods, compounds, compositions and kits useful for delivering to a target site a targeting moiety that is conjugated to one member of a ligand/anti-ligand pair. After localization and clearance of the targeting moiety conjugate, direct or indirect binding of a diagnostic or therapeutic agent conjugate at the target site occurs. Methods for radiometal labeling of biotin or other small molecules, as well as the related compounds, are also disclosed. Background of the Invention Conventional cancer therapy is plagued by two problems. The generally attainable targeting ratio (ratio of administered dose localizing to tumor versus administered dose circulating in blood or ratio of administered dose localizing to tumor versus administered dose migrating to bone marrow) is low. Also, the absolute dose of radiation or therapeutic agent delivered to the tumor is insufficient in many cases to elicit a significant tumor response. Improvement in targeting ratio or absolute dose to tumor is sought. Summary of the Invention The present invention is directed to diagnostic and therapeutic pretargeting methods, moieties useful therein and methods of making those moieties. Such pretargeting methods are characterized by an improved targeting ratio or increased absolute dose to the target cell sites in comparison to conventional cancer therapy. The present invention provides radiolabeled small molecules useful in diagnostic or therapeutic pretargeting methods. Certain embodiments of the present invention include chelate-biotin compounds and conjugates incorporating a chelate and a chemically- modified biotin compound useful in diagnostic or therapeutic pretargeting methods. The present invention further provides methods for making such radiolabeled small molecules. Brief Description of the Drawings Figure 1 illustrates blood clearance of biotinylated antibody following intravenous administration of avidin. Figure 2 depicts radiorhenium tumor uptake in a three-step pretargeting protocol, as compared to administration of radiolabeled antibody (conventional means involving antibody that is covalently linked to chelated radiorhenium) . Figure 3 depicts the tumor uptake profile of NR- LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a control profile of native NR-LU-10 whole antibody. Figure 4 depicts the tumor uptake and blood clearance profiles of NR-LU-10-streptavidin conjugate. Figure 5 depicts the rapid clearance from the blood of asialoorosomucoid in comparison with orosomucoid in terms of percent injected dose of I- 125-labeled protein. Figure 6 depicts the 5 minute limited biodistribution of asialoorosomucoid in comparison with orosomucoid in terms of percent injected dose of I-125-labeled protein. Figure 7 depicts NR-LU-10-streptavidin conjugate blood clearance upon administration of three controls ( # 0) and two doses of a clearing agent at 25 hours post-conjugate administration. Figure 8 shows limited biodistribution data for LU-10-StrAv conjugate upon administration of three controls (Groups 1, 2 and 5) and two doses of clearing agent (Groups 3 and 4) at two hours post-clearing agent administration. Figure 9 depicts NR-LU-10-streptavidin conjugate serum biotin binding capability at 2 hours post- clearing agent administration. Figure 10 depicts NR-LU-10-streptavidin conjugate blood clearance over time upon administration of a control (O ) and three doses of a clearing agent (-^7 , A D) at 24 hours post-conjugate administration. Figure 11A depicts the blood clearance of LU-10- StrAv conjugate upon administration of a control (PBS) and three doses (50, 20 and 10 μg) of clearing agent at two hours post-clearing agent administration. Figure 11B depicts LU-10-StrAv conjugate serum biotin binding capability upon administration of a control (PBS) and three doses (50, 20 and 10 μg) of clearing agent at two hours post-clearing agent administration. Figure 12 depicts a synthetic triamide route to nitro-benzy1-DOTA. Figure 13 depicts a synthetic procedure for the synthesis of p-nitrobenzyl-tetraazacyclododecane via direct peptide cyclization. Figure 14 is an alternative synthetic procedure for the synthesis of p-nitrobenzyl- tetraazacyclododecane via direct peptide cyclization. Figure 15 depicts a synthetic procedure for the synthesis of p-nitrobenzyl-tetraazacyclododecane via reverse direct peptide cyclization. Detailed Description of the Invention Prior to setting forth the invention, it may be helpful to set forth definitions of certain terms to be used within the disclosure. Targeting moiety; A molecule that binds to a defined population of cells. The targeting moiety may bind a receptor, an oligonucleotide, an enzymatic substrate, an antigenic determinant, or other binding site present on or in the target cell population. Antibody is used throughout the specification as a prototypical example of a targeting moiety. Tumor is used as a prototypical example of a target in describing the present invention. Ligand/anti-ligand pair: A complementary/anti- complementary set of molecules that demonstrate specific binding, generally of relatively high affinity. Exemplary ligand/anti-ligand pairs include zinc finger protein/dsDNA fragment, enzyme/inhibitor, hapten/antibody, lectin/carbohydrate, ligand/receptor, and biotin/avidin. Biotin/avidin is used throughout the specification as a prototypical example of a ligand/anti-ligand pair. Anti-ligand: As defined herein, an "anti-ligand" demonstrates high affinity, and preferably, multivalent binding of the complementary ligand. Preferably, the anti-ligand is large enough to avoid rapid renal clearance, and contains sufficient multivalency to accomplish crosslinking and aggregation of targeting moiety-ligand conjugates. Univalent anti-ligands are also contemplated by the present invention. Anti-ligands of the present invention may exhibit or be derivitized to exhibit structural features that direct the uptake thereof, e.g. , galactose residues that direct liver uptake. Avidin and streptavidin are used herein as prototypical anti-ligands. , Avidin: As defined herein, "avidin" includes 5 avidin, streptavidin and derivatives and analogs thereof that are capable of high affinity, multivalent or univalent binding of biotin. Ligand: As defined herein, a "ligand" is a relatively small, soluble molecule that exhibits rapid 10 serum, blood and/or whole body clearance when administered intravenously in an animal or human. Biotin is used as the prototypical ligand. Active Agent: A diagnostic or therapeutic agent ("the payload") , including radionuclides, drugs, anti- 15 tumor agents, toxins and the like. Radionuclide therapeutic agents are used as prototypical active agents. N,.S., Chelates: As defined herein, the term "N x S y chelates" includes bifunctional chelators that are 20 capable of (i) coordinately binding a metal or radiometal and (ii) covalently attaching to a targeting moiety, ligand or anti-ligand. Particularly preferred N x S y chelates have N 2 S 2 and N 3 S cores. Exemplary N x S y chelates are described in Fritzberg et 25 al., Proc. Natl. Acad. Sci. USA 5:4024-29, 1988; in Weber et al., Bioconή . Chem. .1:431-37, 1990; and in the references cited therein, for instance. Pretargeting: As defined herein, pretargeting involves target site localization of a targeting 30 moiety that is conjugated with one member of a ligand/anti-ligand pair; after a time period * sufficient for optimal target-to-non-target accumulation of this targeting moiety conjugate, active agent conjugated to the opposite member of the 35 ligand/anti-ligand pair is administered and is bound (directly or indirectly) to the targeting moiety conjugate at the target site (two-step pretargeting) . Three-step and other related methods described herein are also encompassed. Clearing Agent: An agent capable of binding, complexing or otherwise associating with an administered moiety (e.g. , targeting moiety-ligand, targeting moiety-anti-ligand or anti-ligand alone) present in the recipient's circulation, thereby facilitating circulating moiety clearance from the recipient's body, removal from blood circulation, or inactivation thereof in circulation. The clearing agent is preferably characterized by physical properties, such as size, charge, configuration or a combination thereof, that limit clearing agent access to the population of target cells recognized by a targeting moiety used in the same treatment protocol as the clearing agent. Conjugate: A conjugate encompasses chemical conjugates (covalently or non-covalently bound) , fusion proteins and the like. A recognized disadvantage associated with .in vivo administration of targeting moiety-radioisotopic conjugates for imaging or therapy is localization of the attached radioactive agent at both non-target and target sites. Until the administered radiolabeled conjugate clears from the circulation, normal organs and tissues are transitorily exposed to the attached radioactive agent. For instance, radiolabeled whole antibodies that are administered .in vivo exhibit relatively slow blood clearance; maximum target site localization generally occurs 1-3 days post- administration. Generally, the longer the clearance time of the conjugate from the circulation, the greater the radioexposure of non-target organs. These characteristics are particularly problematic with human radioimmunotherapy. In human clinical trials, the long circulating half-life of radioisotope bound to whole antibody causes relatively large doses of radiation to be delivered to the whole body. In particular, the bone marrow, which is very radiosensitive, is the dose-limiting organ of non- specific toxicity. In order to decrease radioisotope exposure of non- target tissue, potential targeting moieties generally have been screened to identify those that display minimal non-target reactivity, while retaining target specificity and reactivity. By reducing non-target exposure (and adverse non-target localization and/or toxicity) , increased doses of a radiotherapeutic conjugate may be administered; moreover, decreased non-target accumulation of a radiodiagnostic conjugate leads to improved contrast between background and target. Therapeutic drugs, administered alone or as targeted conjugates, are accompanied by similar disadvantages. Again, the goal is administration of the highest possible concentration of drug (to maximize exposure of target tissue) , while remaining below the threshold of unacceptable normal organ toxicity (due to non-target tissue exposure) . Unlike radioisotopes, however, therapeutic drugs need to be taken into a target cell to exert a cytotoxic effect. In the case of targeting moiety-therapeutic drug conjugates, it would be advantageous to combine the relative target specificity of a targeting moiety with a means for enhanced target cell internalization of the targeting moiety-drug conjugate. In contrast, enhanced target cell internalization is disadvantageous if one administers diagnostic agent-targeting moiety conjugates. Internalization of diagnostic conjugates results in cellular catabolism and degradation of the conjugate. Upon degradation, small adducts of the diagnostic agent or the diagnostic agent per se may be released from the cell, thus eliminating the ability to detect the conjugate in a target-specific manner. One method for reducing non-target tissue exposure to a diagnostic or therapeutic agent involves "pretargeting" the targeting moiety at a target site, and then subsequently administering a rapidly clearing diagnostic or therapeutic agent conjugate that is capable of binding to the "pretargeted" targeting moiety at the target site. A description of some embodiments of the pretargeting technique may be found in US Patent No. 4,863,713 (Goodwin et al.) . A typical pretargeting approach ("three-step") is schematically depicted below. j| Targeting moiety ie Anti-ligand 0 Ligand ffl Ligand-active agent site (i.e., receptor, antigenic determinant) three-step pretargeting protocol features administration of an antibody-ligand conjugate, which is allowed to localize at a target site and to dilute in the circulation. Subsequently administered anti-ligand binds to the antibody-ligand conjugate and clears unbound antibody-ligand conjugate from the blood. Preferred anti-ligands are large and contain sufficient multivalency to accomplish crosslinking and aggregation of circulating antibody- ligand conjugates. The clearing by anti-ligand is probably attributable to anti-ligand crosslinking and/or aggregation of antibody-ligand conjugates that are circulating in the blood, which leads to complex/aggregate clearance by the recipient's RES (reticuloendothelial system) . Anti-ligand clearance of this type is preferably accomplished with a multivalent molecule; however, a univalent molecule of sufficient size to be cleared by the RES on its own could also be employed. Alternatively, receptor-based clearance mechanisms, e.g. , Ashwell receptor or hexose residue, such as galactose or mannose residue, recognition mechanisms, may be responsible for anti- ligand clearance. Such clearance mechanisms are less dependent upon the valency of the anti-ligand with respect to the ligand than the RES complex/aggregate clearance mechanisms. It is preferred that the ligand-anti-ligand pair displays relatively high affinity binding. A diagnostic or therapeutic agent-ligand conjugate that exhibits rapid whole body clearance is then administered. When the circulation brings the active agent-ligand conjugate in proximity to the target cell-bound antibody-ligand-anti-ligand complex, anti- ligand binds the circulating active agent-ligand conjugate and produces an antibody-ligand : anti- ligand : ligand-active agent "sandwich" at the target site. Because the diagnostic or therapeutic agent is attached to a rapidly clearing ligand (rather than antibody, antibody fragment or other slowly clearing targeting moiety) , this technique promises decreased non-target exposure to the active agent. Alternate pretargeting methods eliminate the step of parenterally administering an anti-ligand clearing agent. These "two-step" procedures feature targeting moiety-ligand or targeting moiety-anti-ligand administration, followed by administration of active agent conjugated to the opposite member of the ligand- anti-ligand pair. As an optional step "1.5" in the two-step pretargeting methods of the present invention, a clearing agent (preferably other than ligand or anti-ligand alone) is administered to facilitate the clearance of circulating targeting moiety-containing conjugate. In the two-step pretargeting approach, the clearing agent preferably does not become bound to the target cell population, either directly or through the previously administered and target cell bound targeting moiety-anti-ligand or targeting moiety- ligand conjugate. An example of two-step pretargeting involves the use of biotinylated human transferrin as a clearing agent for avidin-targeting moiety conjugate, wherein the size of the clearing agent results in liver clearance of transferrin-biotin- circulating avidin-targeting moiety complexes and substantially precludes association with the avidin- targeting moiety conjugates bound at target cell sites. (See, Goodwin, D.A., Antibod. Immunocon . Radiopharm. , 4.: 427-34, 1991). The two-step pretargeting approach overcomes certain disadvantages associated with the use of a clearing agent in a three-step pretargeted protocol. More specifically, data obtained in animal models demonstrate that in vivo anti-ligand binding to a pretargeted targeting moiety-ligand conjugate (i.e., the cell-bound conjugate) removes the targeting moiety-ligand conjugate from the target cell. One explanation for the observed phenomenon is that the multivalent anti-ligand crosslinks targeting moiety- ligand conjugates on the cell surface, thereby initiating or facilitating internalization of the resultant complex. The apparent loss of targeting moiety-ligand from the cell might result from internal degradation of the conjugate and/or release of active agent from the conjugate (either at the cell surface or intracellularly) . An alternative explanation for the observed phenomenon is that permeability changes in the target cell's membrane allow increased passive diffusion of any molecule into the target cell. Also, some loss of targeting moiety-ligand may result from alteration in the affinity by subsequent binding of another moiety to the targeting moiety-ligand, e.g. , anti-idiotype monoclonal antibody binding causes removal of tumor bound monoclonal antibody. The present invention recognizes that this phenomenon (apparent loss of the targeting moiety- ligand from the target cell) may be used to advantage with regard to .in vivo delivery of therapeutic agents generally, or to drug delivery in particular. For instance, a targeting moiety may be covalently linked to both ligand and therapeutic agent and administered to a recipient. Subsequent administration of anti- ligand crosslinks targeting moiety-ligand-therapeutic agent tripartite conjugates bound at the surface, inducing internalization of the tripartite conjugate (and thus the active agent) . Alternatively, targeting moiety-ligand may be delivered to the target cell surface, followed by administration of anti-ligand- therapeutic agent. In one aspect of the present invention, a targeting moiety-anti-ligand conjugate is administered in vivo; upon target localization of the targeting moiety-anti-ligand conjugate (i.e., and clearance of this conjugate from the circulation) , an active agent- ligand conjugate is parenterally administered. This method enhances retention of the targeting moiety- anti-ligand : ligand-active agent complex at the target cell (as compared with targeting moiety-ligand : anti-ligand : ligand-active agent complexes and targeting moiety-ligand : anti-ligand-active agent complexes) . Although a variety of ligand/anti-ligand pairs may be suitable for use within the claimed invention, a preferred ligand/anti-ligand pair is biotin/avidin. In a second aspect of the invention, radioiodinated biotin and related methods are disclosed. Previously, radioiodinated biotin derivatives were of high molecular weight and were difficult to characterize. The radioiodinated biotin described herein is a low molecular weight compound that has been easily and well characterized. In a third aspect of the invention, a targeting moiety-ligand conjugate is administered in vivo; upon target localization of the targeting moiety-ligand conjugate (i.e., and clearance of this conjugate from the circulation) , a drug-anti-ligand conjugate is parenterally administered. This two-step method not only provides pretargeting of the targeting moiety conjugate, but also induces internalization of the subsequent targeting moiety-ligand-anti-ligand-drug complex within the target cell. Alternatively, another embodiment provides a three-step protocol that produces a targeting moiety-ligand : anti-ligand : ligand-drug complex at the surface, wherein the ligand-drug conjugate is administered simultaneously or within a short period of time after administration of anti-ligand (i.e., before the targeting moiety- ligand-anti-ligand complex has been removed from the target cell surface) . In a fourth aspect of the invention, methods for radiolabeling biotin with technetium-99m, rhenium-186 and rhenium-188 are disclosed. Previously, biotin derivatives were radiolabeled with indium-Ill for use in pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl. Med. Biol. 18:719-26. 1991; Kalofonos et al., J. Nucl. Med. 31: 1791-96, 1990; Paganelli et al., Cane. Res. .51:5960-66, 1991). However, ""Tc is a particularly preferred radionuclide for immunoscintigraphy due to (i) low cost, (ii) convenient supply and (iii) favorable nuclear properties. Rhenium-186 displays chelating chemistry very similar to "Tc, and is considered to be an excellent therapeutic radionuclide (i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similar to 131 I) . Therefore, the claimed methods for technetium and rhenium radiolabeling of biotin provide numerous advantages. The "targeting moiety" of the present invention binds to a defined target cell population, such as tumor cells. Preferred targeting moieties useful in this regard include antibody and antibody fragments, peptides, and hormones. Proteins corresponding to known cell surface receptors (including low density lipoproteins, transferrin and insulin) , fibrinolytic enzymes, anti-HER2, platelet binding proteins such as annexins, and biological response modifiers (including interleukin, interferon, erythropoietin and colony- stimulating factor) are also preferred targeting moieties. Also, anti-EGF receptor antibodies, which internalize following binding to the receptor and traffic to the nucleus to an extent, are preferred targeting moieties for use in the present invention to facilitate delivery of Auger emitters and nucleus binding drugs to target cell nuclei. Oligonucleotides, e.g. , antisense oligonucleotides that are complementary to portions of target cell nucleic acids (DNA or RNA) , are also useful as targeting moieties in the practice of the present invention. Oligonucleotides binding to cell surfaces are also useful. Analogs of the above-listed targeting moieties that retain the capacity to bind to a defined target cell population may also be used within the claimed invention. In addition, synthetic targeting moieties may be designed. Functional equivalents of the aforementioned molecules are also useful as targeting moieties of the present invention. One targeting moiety functional equivalent is a "mimetic" compound, an organic chemical construct designed to mimic the proper configuration and/or orientation for targeting moiety- target cell binding. Another targeting moiety functional equivalent is a short polypeptide designated as a "minimal" polypeptide, constructed using computer-assisted molecular modeling and mutants having altered binding affinity, which minimal polypeptides exhibit the binding affinity of the targeting moiety. Preferred targeting moieties of the present invention are antibodies (polyclonal or monoclonal) , peptides, oligonucleotides or the like. Polyclonal antibodies useful in the practice of the present invention are polyclonal (Vial and Callahan, Univ. Mich. Med. Bull. , 20: 284-6, 1956), affinity-purified polyclonal or fragments thereof (Chao et al., Res. Comm. in Che . Path. & Pharm.. _9_: 749-61, 1974) . Monoclonal antibodies useful in the practice of the present invention include whole antibody and fragments thereof. Such monoclonal antibodies and fragments are producible in accordance with conventional techniques, such as hybridoma synthesis, recombinant DNA techniques and protein synthesis. Useful monoclonal antibodies and fragments may be derived from any species (including humans) or may be formed as chimeric proteins which employ sequences from more than one species. See, generally, Kohler and Milstein, Nature. 256: 495-97, 1975; Eur. J. Immunol.. _6 : 511-19, 1976. Human monoclonal antibodies or "humanized" murine antibody are also useful as targeting moieties in accordance with the present invention. For example, murine monoclonal antibody may be "humanized" by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e.. containing the antigen binding sites) or the complementarity determining regions thereof with the nucleotide sequence encoding a human constant domain region and an Fc region, e.g.. in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2. Some murine residues may also be retained within the human variable region framework domains to ensure proper target site binding characteristics. Humanized targeting moieties are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half-life and a reduction in the possibility of adverse immune reactions. Types of active agents (diagnostic or therapeutic) useful herein include toxins, anti-tumor agents, drugs and radionuclides. Several of the potent toxins useful within the present invention consist of an A and a B chain. The A chain is the cytotoxic portion and the B chain is the receptor-binding portion of the intact toxin molecule (holotoxin) . Because toxin B chain may mediate non-target cell binding, it is often advantageous to conjugate only the toxin A chain to a targeting protein. However, while elimination of the toxin B chain decreases non-specific cytotoxicity, it also generally leads to decreased potency of the toxin A chain-targeting protein conjugate, as compared to the corresponding holotoxin-targeting protein conjugate. Preferred toxins in this regard include holotoxins, such as abrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussis toxin and Shiga toxin; and A chain or "A chain-like" molecules, such as ricin A chain, abrin A chain, modeccin A chain, the enzymatic portion of Pseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic portion of pertussis toxin, the enzymatic portion of Shiga toxin, gelonin, pokeweed antiviral protein, saporin, tritin, barley toxin and snake venom peptides. Ribosomal inactivating proteins (RIPs) , naturally occurring protein synthesis inhibitors that lack translocating and cell-binding ability, are also suitable for use herein. Extremely highly toxic toxins, such as palytoxin and the like, are also contemplated for use in the practice of the present invention. Preferred drugs suitable for use herein include conventional chemotherapeutics, such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide and cis- platinum, as well as other conventional chemotherapeutics as described in Cancer: Principles and Practice of Oncology. 2d ed. , V.T. DeVita, Jr., S. Hellman, S.A. Rosenberg, J.B. Lippincott Co., Philadelphia, PA, 1985, Chapter 14. A particularly preferred drug within the present invention is a trichothecene. Trichothecenes are drugs produced by soil fungi of the class Fungi imperfecti or isolated from Baccharus megapot arnica (Bamburg, J.R. Proc. Molec. Subcell. Biol. 8.:41-110, 1983; Jarvis & Mazzola, Ace. Chem. Res. r5:338-395, 1982). They appear to be the most toxic molecules that contain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org. Naturst. H:61-117, 1974). They are all reported to act at the level of the ribosome as inhibitors of protein synthesis at the initiation, elongation, or termination phases. There are two broad classes of trichothecenes: those that have only a central sesquiterpenoid structure and those that have an additional macrocyclic ring (simple and macrocyclic trichothecenes, respectively) . The simple trichothecenes may be subdivided into three groups (i.e., Group A, B, and C) as described in U.S. Patent Nos. 4,744,981 and 4,906,452 (incorporated herein by reference) . Representative examples of Group A simple trichothecenes include: Scirpene, Roridin C, dihydrotrichothecene, Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2 tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin, deacetylcalonectrin, calonectrin, diacetylverrucarol, 4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol, 7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol) , 7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol, 8-acetylneosolaniol, NT-1, NT-2, HT-2, T-2, and acetyl T-2 toxin. Representative examples of Group B simple trichothecenes include: Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, 3, 15-diacetyldeoxynivalenol, Nivalenol, 4-acetylnivalenol (Fusarenon-X) , 4, 15-idacetylnivalenol, 4,7, 15-triacetylnivalenol, and tetra-acetylnivalenol. Representative examples of Group C simple trichothecenes include: Crotocol and Crotocin. Representative macrocyclic trichothecenes include Verrucarin A, Verrucarin B, Verrucarin J (Satratoxin C) , Roridin A, Roridin D, Roridin E (Satratoxin D) , Roridin H, Satratoxin F, Satratoxin G, Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A, Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, and Roritoxin D. In addition, the general "trichothecene" sesquiterpenoid ring structure is also present in compounds termed "baccharins" isolated from the higher plant Baccharis megapotamica, and these are described in the literature, for instance as disclosed by Jarvis et al. (Chemistry of Alleopathy, ACS Symposium Series No. 268: ed. A.C. Thompson, 1984, pp. 149-159). Experimental drugs, such as ercaptopurine, N- methylformamide, 2-amino-l,3,4-thiadiazole, melphalan, hexamethylmelamine, gallium nitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin, bromodeoxyuridine, iododeoxyuridine, semustine, l-(2- chloroethyl) -3-(2, 6-dioxo-3-piperidyl) -1-nitrosourea, N,N'-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwinia asparaginase, ifosfamide, 2- mercaptoethane sulfonate, teniposide, taxol, 3- deazauridine, soluble Baker's antifol, homoharringtonine, cyclocytidine, acivicin, ICRF-187, spiromustine, levamisole, chlorozotocin, aziridinyl benzoquinone, spirogermanium, aclarubicin, pentostatin, PALA, carboplatin, amsacrine, caracemide, iproplatin, isonidazole, dihydro-5-azacytidine, 4'- deoxy-doxorubicin, menogaril, triciribine phosphate, fazarabine, tiazofurin, teroxirone, ethiofos, N-(2- hydroxyethyl) -2-nitro-lH-imidazole-l-acetamide, mitoxantrone, acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B, merbarone, dihydrolenperone, flavone-8-acetic acid, oxantrazole, ipomeanol, tri etrexate, deoxyspergualin, echinomycin, and dideoxycytidine (see NCI Investigational Drugs, Pharmaceutical Data 1987, NIH Publication No. 88-2141, Revised November 1987) are also preferred. Radionuclides useful within the present invention include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence- emitters, with beta- or alpha-emitters preferred for therapeutic use. Radionuclides are well-known in the art and include 123 I, 125 I, 130 I, 131 I, 133 I, 135 I, 7 Sc, ^As, 72 Se, 90 Y, 88 Y, "Ru, 100 Pd, 101m Rh, 119 Sb, 128 Ba, 197 Hg, 211 At, 212 Bi, 153 Sm, 169 Eu, 212 Pb, 109 Pd, ιn In, 67 Ga, 68 Ga, 67 Cu, 75 Br, 76 Br, "Br, """Tc, n C, 13 N, 15 0 and I8 F. Preferred therapeutic radionuclides include 188 Re, 186 Re, 203 Pb, 212 Pb, 212 Bi, 109 Pd, «Cu, 67 Cu, 90 Y, 125 I, 131 I, π Br, 2n At, ^Ru, 10S Rh, 198 Au and 199 Ag or 177 Lu. Other anti-tumor agents.e.g. , agents active against proliferating cells, are administrable in accordance with the present invention. Exemplary anti-tumor agents include cytokines, such as IL-2, tumor necrosis factor or the like, lectin inflammatory response promoters (selectins) , such as L-selectin, E- selectin, P-selectin or the like, and like molecules. Ligands suitable for use within the present invention include biotin, haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitors and analogs and derivatives thereof. Useful complementary anti- ligands include avidin (for biotin) , carbohydrates (for lectins) and antibody, fragments or analogs thereof, including mimetics (for haptens and epitopes) and zinc finger proteins (for dsDNA fragments) and enzymes (for enzyme inhibitors) . Preferred ligands and anti-ligands bind to each other with an affinity of at least about k D > 10 9 M. The 1,4,7, 10-tetraazacyclododecane-N,N' ,N",N'"- tetra acetic acid (DOTA) -biotin conjugate (DOTA-LC- biotin) depicted below has been reported to have desirable in vivo biodistribution and is cleared primarily by renal excretion. DOTA may also be con ugated to other l gands or to 0 anti-ligands in the practice of the present invention. Because DOTA strongly binds Y-90 and other radionuclides, it has been proposed for use in radioimmunotherapy. For therapy, it is very important that the radionuclide be stably bound within the DOTA chelate and that the DOTA chelate be stably attached to a ligand or anti-ligand. For illustrative purposes, DOTA-biotin conjugates are described. Only radiolabeled DOTA-biotin conjugates exhibiting those two characteristics are useful to deliver radionuclides to the targets. Release of the radionuclide from the DOTA chelate or cleavage of the biotin and DOTA conjugate components in serum or at non-target sites renders the conjugate unsuitable for use in therapy. Serum stability of DOTA-LC-biotin (where LC refers to the "iong chain" linker, including an aminocaproyl spacer between the biotin and the DOTA conjugate components) shown above, while reported in the literature to be good, has proven to be problematic. Experimentation has revealed that DOTA-LC-biotin is rapidly cleared from the blood and excreted into the urine as fragments, wherein the biotinamide bond rather than the DOTA-amide bond has been cleaved, as shown below. conjugates produced parallel results as shown below. Cleavage of the benza ide was not observed as evidenced by the absence of detectable quantities of iodobenzoic acid in the serum. It appears that the cleavage results from the action of serum biotinidase. Biotinidase is a hydrolytic enzyme that catalyzes the cleavage of biotin from biotinyl peptides. See, for example, Evangelatos, et al., "Biotinidase Radioassay Using an I-125-Biotin Derivative, Avidin, and Polyethylene Glycol Reagents," Analytical Biochemistry. 196: 385- 89, 1991. Drug-biotin conjugates which structurally resemble biotinyl peptides are potential substrates for cleavage by plasma biotinidase. Poor in vivo stability therefore limits the use of drug-biotin conjugates in therapeutic applications. The use of peptide surrogates to overcome poor stability of peptide therapeutic agents has been an area of intense research effort. See, for example, Spatola, Peptide Backbone Modification: A Structure-Activity Analysis of Peptide Containing Amide Bond Surrogates,, "Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, " vol. 7, Weinstein, ed. , Marcel Dekker, New York, 1983; and Kim et al., "A New Peptide Bond Surrogate: 2-Isoxazoline in Pseudodipeptide Chemistry," Tetrahedron Letters, 45: 6811-14, 1991. Elimination of the aminocaproyl spacer of DOTA-LC- biotin gives DOTA-SC-biotin (where the SC indicates the "short chain" linker between the DOTA and biotin conjugate components) , which molecule is shown below: DOTA-SC-biotin exhibits significantly improved serum stability in comparison to DOTA-LC-biotin. This result does not appear to be explainable on the basis of biotinidase activity alone. The experimentation leading to this conclusion is summarized in the Table set forth below. Time Dependent Cleavage of DOTA-Biotin Conjugates Time at 37 'C % Avidin Binding PIP-Biocytin Y-90-LC Y-90-SC s DOTA-Biotin DOTA-Biotin 98% 60% where "-" indicates that the value was not measured. The difference in serum stability between DOTA-LC- biotin and DOTA-SC-biotin might be explained by the fact that the SC derivative contains an aromatic amide linkage in contrast to the aliphatic amide linkage of the LC derivative, with the aliphatic amide linkage being more readily recognized by enzymes as a substrate therefor. This argument cannot apply to biotinidase, however, because biotinidase very efficiently cleaves aromatic amides. In fact, it is recognized that the simplest and most commonly employed biotinidase activity measuring method uses N- (d-biotinyl)-4-aminobenzoate (BPABA) as a substrate, with the hydrolysis of BPABA resulting in the liberation of biotin and 4-aminobenzoate (PABA) . See, for example, B. Wolf, et al. , "Methods in Enzymology," pp. 103-111, Academic Press Inc., 1990. Consequently, one would predict that DOTA-SC-biotin, like its LC counterpart, would be a biotinidase substrate. Since DOTA-SC-biotin exhibits serum stability, biotinidase activity alone does not adequately explain why some conjugates are serum stable while others are not. A series of DOTA-biotin conjugates was therefore synthesized by the present inventors to determine which structural features conferred serum stability to the conjugates. Some general strategies for improving serum stability of peptides with respect to enzymatic action are the following: incorporation of D-amino acids, N- ethyl amino acids and alpha-substituted amino acids. In vivo stable biotin-DOTA conjugates are useful within the practice of the present invention. In vivo stability imparts the following advantages: 1) increased tumor uptake in that more of the radioisotope will be targeted to the previously localized targeting moiety-streptavidin; and 2) increased tumor retention, if biotin is more stably bound to the radioisotope. In addition, the linkage between DOTA and biotin may also have a significant impact on biodistribution (including normal organ uptake, target uptake and the like) and pharmacokinetics. The strategy for design of the DOTA-containing molecules and conjugates of the present invention involved three primary considerations: 1) in vivo stability (including biotinidase and general peptidase activity resistance) , with an initial acceptance criterion of 100% stability for 1 hour; 2) renal excretion; and 3) ease of synthesis. The DOTA-biotin conjugates of the present invention reflect the implementation of one or more of the following strategies: 1) substitution of the carbon adjacent to the cleavage susceptible amide nitrogen; 2) alkylation of the cleavage susceptible amide nitrogen; 3) substitution of the amide carbonyl with an alkyl amino group; 4) incorporation of D-amino acids as well as analogs or derivatives thereof; or 5) incorporation of thiourea linkages. DOTA-biotin conjugates in accordance with the present invention may be generally characterized as follows: conjugates that retain the biotin carboxy group in the structure thereof and those that do not (i.e. , the terminal carboxy group of biotin has been reduced or otherwise chemically modified. Structures of such conjugates represented by the following general formula have been devised: wherein L may alternatively be substituted in one of the following ways on one of the -CH 2 -COOH branches of the DOTA structure: -CH(L)-COOH or -CH 2 COOL or -CH 2 COL) . When these alternative structures are employed, the portion of the linker bearing the functional group for binding with the DOTA conjugate component is selected for the capability to interact with either the carbon or the carboxy in the branch portions of the DOTA structure, with the serum stability conferring portion of the linker structure being selected as described below. In the case where the linkage is formed on the core of the DOTA structure as shown above, L is selected according to the following principles, with the portion of the linker designed to bind to the DOTA conjugate component selected for the capability to bind to an amine. A. One embodiment of the present invention includes linkers incorporating a D-amino acid spacer between a DOTA aniline amine and the biotin carboxy group shown above. Substituted amino acids are preferred for these embodiments of the present invention, because alpha-substitution also confers enzymatic cleavage resistance. Exemplary L moieties, of this embodiment of the present invention may be represented as follows: where R 1 is selected from lower alkyl, lower alkyl substituted with hydrophilic groups (preferably, (CH 2 ) n -OH, (CH 2 ) n -OS0 3 , (CH 2 ) n -S0 3 , , wh(≥re n is 1 or 2) , glucuronide-substituted amino acids or other glucuronide derivatives; and R 2 is selected from hydrogen, lower alkyl, substituted lower alkyl (e.g. , hydroxy, sulfate, phosphonate or a hydrophilic moiety (preferably OH) . For the purposes of the present disclosure, the term "lower alkyl" indicates an alkyl group with from one to five carbon atoms. Also, the term "substituted" includes one or several substituent groups, with a single substituent group preferred. Preferred L groups of this embodiment of the present invention include the following: R 1 = CH 3 and R 2 = H (a D-alanine derivative, with a synthetic scheme therefor shown in Example XV) ; R 1 = CH 3 and R 2 = CH 3 (an N-methyl-D-alanine derivative) ; R 1 = CH 2 -OH and R 2 = H (a D-serine derivative) ; R 1 = CH 2 OS0 3 and R 2 = H (a D-serine-O-sulfate- derivative) ; and OH R- = CH 2 0P0 lH and R ~ = H (a D-serme-O-phosphonate- 0 derivative) ; Other preferred moieties of this embodiment of the present invention include molecules wherein R 1 is hydrogen and R 2 = -(CH 2 ) n 0H or a sulfate or phosphonate derivative thereof and n is 1 or 2 as well as molecules wherein R 1 is Preferred moieties incorporating the glucuronide of D-lysine and the glucuronide of amino pimelate are shown below as I and II, respectively. A particularly preferred linker of this embodiment of the present invention is the D-alanine derivative set forth above. B. Linkers incorporating alkyl substitution on one or more amide nitrogen atoms are also encompassed by the present invention, with some embodiments of such linkers preparable from L-amino acids. Amide bonds having a substituted amine moiety are less susceptible to enzymatic cleavage. Such linkers exhibit the following general formula: where R 4 is selected from hydrogen, lower a__.kyl, lower alkyl substituted with hydroxy, sulfate, phosphonate R 3 is selected from hydrogen; an amine; lower alkyl; an amino- or a hydroxy-, sulfate- or phosphonate-substituted lower alkyl; a glucuronide or a glucuronide-derivatized amino groups; and n ranges from 0-4. Preferred linkers of this embodiment of the present invention include: R 3 = H and R 4 = CH 3 when n = 4, synthesizable as discussed in Example XV; R 3 = H and R 4 = CH 3 when n = 0, synthesizable from N-methyl-glycine (having a trivial name of εarcosine) as described in Example XV; R 3 = R 3 = when n = 4 (Bis-DOTA-LC-biotin) , synthesizable from bromohexanoic acid as discussed in Example XV; and n = 0 (bis-DOTA-SC-biotin) , synthesizable from iminodiacetic acid. The synthesis of a conjugate including a linker wherein R 3 is H and R 4 is -CH 2 CH 2 OH and n is 0 is also described in Example XV. Schematically, the synthesis of a conjugate of this embodiment of the present invention wherein n is 0, R 3 is H and R 4 is -CH 2 -COOH is shown below. .) NaH 0 O II II 2) t -butyl-0-C-CH 2 -Br CHj-C-O-t-butyl NaOH HO-C-CH2-NH-C-CF 3 HO-C-CHj-N-C-CF j 0 Bis-DOTA-LC-biotin, for example, offers the following advantages: 1) incorporation of two DOTA molecules on one biotin moiety increases the overall hydrophilicity of the biotin conjugate and thereby directs in vivo distribution to urinary excretion; and 2) substitution of the amide nitrogen adjacent to the biotin carboxyl group blocks peptide and/or biotinidase cleavage at that site. Bis-DOTA-LC-biotin, the glycine-based linker and the N-methylated linker where R 3 = H, R 4 = CH 3 , n = 4 are particularly preferred linkers of this embodiment of the present invention. C. Another linker embodiment incorporates a thiourea moiety therein. Exemplary thiourea adducts of the present invention exhibit the following general formula: where R 5 is selected from hydrogen or lower alkyl; R 6 is selected from H and a hydrophilic moiety; and n ranges from 0-4. Preferred linkers of this embodiment of the present invention are as follows: R 5 = H and R 6 = H when n = 5; R 5 = H and R 6 = COOH when n = 5; and R 5 = CH 3 and R 6 = COOH when n = 5. The second preferred linker recited above can be prepared using either L-lysine or D-lysine. Similarly, the third preferred linker can be prepared using either N-methyl-D-lysine or N-methyl-L-lysine. Another thiourea adduct of minimized lipophilicity is which may be formed via the addition of biotinhydrazide (commercially available from Sigma Chemical Co., St. Louis, Missouri) and DOTA-benzyl- isothiocyanate (a known compound synthesized in one step from DOTA-aniline) , with the thiourea-containing compound formed as shown below. D. Amino acid-derived linkers of the present invention with substitution of the carbon adjacent to the cleavage susceptible amide have the general formula set forth below: wherein Z is -(CH 2 ) 2 -, conveniently synthesized form glutamic acid; or Z = -CH 2 -S-CH 2 -, synthesizable from cysteine and iodo-acetic acid; or Z = -CH 2 -, conveniently synthesized form aspartic acid; or Z = -(CH 2 ) n -CO-0-CH 2 -, where n ranges from 1-4 and which is synthesizable from serine. E. Another exemplary linker embodiment of the present invention has the general formula set forth below: and n ranges from 1-5. F. Another embodiment involves disulfide- containing linkers, which provide a metabolically cleavable moiety (-S-S-) to reduce non-target retention of the biotin-DOTA conjugate. Exemplary linkers of this type exhibit the following formula: —CH? —( )/—NH-CO-fCH,Jn-S-S-fCHj) n —NH-CO— where n and n' preferably range between 0 and 5. The advantage of using conditionally cleavable linkers is an improvement in target/non-target localization of the active agent. Conditionally cleavable linkers include enzymatically cleavable linkers, linkers that are cleaved under acidic conditions, linkers that are cleaved under basic conditions and the like. More specifically, use of linkers that are cleaved by enzymes, which are present in non-target tissues but reduced in amount or absent in target tissue, can increase target cell retention of active agent relative to non-target cell retention. Such conditionally cleavable linkers are useful, for example, in delivering therapeutic radionuclides to target cells, because such active agents do not require internalization for efficacy, provided that the linker is stable at the target cell surface or protected from target cell degradation. Cleavable linkers are also useful to effect target site selective release of active agent at target sites. Active agents that are preferred for cleavable linker embodiments of the present invention are those that are substantially non-cytotoxic when conjugated to ligand or anti-ligand. Such active agents therefore require release from the ligand- or anti- ligand-containing conjugate to gain full potency. For example, such active agents, while conjugated, may be unable to bind to a cell surface receptor; unable to internalize either actively or passively; or unable to serve as a binding substrate for a soluble (intra- or inter-cellular) binding protein or enzyme. Exemplary of an active agent-containing conjugate of this type is chemotherapeutic drug-cis-aconityl-biotin. The cis-aconityl linker is acid sensitive. Other acid sensitive linkers useful in cleavable linker embodiments of the present invention include esters, thioesters and the like. Use of conjugates wherein an active agent and a ligand or an anti-ligand are joined by a cleavable linker will result in the selective release of the active agent at tumor cell target sites, for example, because the inter-cellular milieu of tumor tissue is generally of a lower pH (more highly acidic) than the inter-cellular milieu of normal tissue. G. Ether, thioether, ester and thioester linkers are also useful in the practice of the present invention. Ether and thioether linkers are stable to acid and basic conditions and are therefore useful to deliver active agents that are potent in conjugated form, such as radionuclides and the like. Ester and thioesters are hydrolytically cleaved under acidic or basic conditions or are cleavable by enzymes including esterases, and therefore facilitate improved target:non-target retention. Exemplary linkers of this type have the following general formula: where X is O or S; and Q is a bond, a methylene group, a -CO- group or -CO-(CH 2 ) n -NH-; and n ranges from 1-5. Other such linkers have the general formula: -CH 2 -X-Q, where Q and X are defined as set forth above. H. Another amino-containing linker of the present invention is structured as follows: where R 7 is lower alkyl, preferably methyl. In this case, resistance to enzymatic cleavage is conferred by the alkyl substitution on the amine. I. Polymeric linkers are also contemplated by the present invention. Dextran and cyclodextran are preferred polymers useful in this embodiment of the present invention as a result of the hydrophilicity of the polymer, which leads to favorable excretion of conjugates containing the same. Other advantages of using dextran polymers are that such polymers are substantially non-toxic and non-immunogenic, that they are commercially available in a variety of sizes and that they are easy to conjugate to other relevant molecules. Also, dextran-linked conjugates exhibit advantages when non-target sites are accessible to dextranase, an enzyme capable of cleaving dextran polymers into smaller units while non-target sites are not so accessible. Other linkers of the present invention are produced prior to conjugation to DOTA and following the reduction of the biotin carboxy moiety. These linkers of the present invention have the following general formula: Embodiments of linkers of this aspect of the present invention include the following: 34 J. An ether linkage as shown below may be formed in a DOTA-biotin conjugate in accordance with the procedure indicated below. L' = -NH-CO-(CH 2 ) n -0- where n ranges from 1 to 5, with 1 preferred. This linker has only one amide moiety which is bound directly to the DOTA aniline (as in the structure of DOTA-SC-biotin) . In addition, the ether linkage imparts hydrophilicity, an important factor in facilitating renal excretion. K. An amine linker formed from reduced biotin (hydroxybiotin or aminobiotin) is shown below, with conjugates containing such a linker formed, for example, in accordance with the procedure described in Example XV. L ' = -NH- This linker contains no amide moieties and the unalkylated amine may impart favorable biodistribution properties since unalkylated DOTA-aniline displays excellent renal clearance. L. Substituted amine linkers, which can form conjugates via amino-biotin intermediates, are shown below. = -(CH 2 ) q -NH-CO-CH 2 -N-R 9 V where R 8 is H; -(CH 2 ) 2 -OH or a sulfate or phosphonate derivative thereof ; or ; or the like; and R 9 is a bond or -(CH 2 ) n -CO-NH-, where n ranges from 0-5 and is preferably 1 and where q is 0 or 1. These moieties exhibit the advantages of an amide only directly attached to DOTA-aniline and either a non- amide amine imparting a positive charge to the linker in vivo or a N-alkylated glucuronide hydrophilic group, each alternative favoring renal excretion. M. Amino biotin may also be used as an intermediate in the production of conjugates linked by linkers having favorable properties, such as a thiourea-containing linker of the formula: L' = -NH-CS-NH- Conjugates containing this thiourea linker have the following advantages: no cleavable amide and a short, fairly polar linker which favors renal excretion. A bis-DOTA derivative of the following formula can also be formed from amino-biotin. where n ranges from 1 to 5, with 1 and 5 preferred. This molecule offers the advantages of the previously discussed bis-DOTA derivatives with the added advantage of no cleavable amides. Additional linkers of the present invention which are employed in the production of conjugates characterized by a reduced biotin carboxy moiety are the following: L = -(CH 2 ) 4 -NH-, wherein the amine group is attached to the methylene group corresponding to the reduced biotin carboxy moiety and the methylene chain is attached to a core carbon in the DOTA ring. Such a linker is conveniently synthesizable from lysine. L = -(CH 2 ) q -C0-NH-, wherein q is 1 or 2, and wherein the amine group is attached to the methylene group corresponding to the reduced biotin carboxy moiety and the methylene group(s) are attached to a core carbon in the DOTA ring. This moiety is synthesizable from amino-biotin. The linkers set forth above are useful to produce conjugates having one or more of the following advantages: - bind avidin or streptavidin with the same or substantially similar affinity as free biotin; - bind metal M +3 ions efficiently and with high kinetic stability; - are excreted primarily through the kidneys into urine; - are stable to. bodily fluid amidases; - penetrate tissue rapidly and bind to pretargeted avidin or streptavidin; and - are excreted rapidly with a whole body residence half-life of less than about 5 hours. Synthetic routes to an intermediate of the DOTA- biotin conjugates depicted above, nitrobenzyl-DOTA, have been proposed. These proposed synthetic routes produce the intermediate compound in suboptimal yield, however. For example, Renn and Meares, "Large Scale Synthesis of Bifunctional Chelating Agent Q-(p- nitrobenzyl)-1,4,7,10-tetraazacyclododecane- N,N',N' ' ,N' ' '-tetra acetic acid, and the Determination of its Enantiomeric Purity by Chiral Chro atography," Bioconi . Chem. , 3_: 563-9, 1992, describe a nine-step synthesis of nitrobenzyl-DOTA, including reaction steps that either proceed in low yield or involve cumbersome transformations or purifications. More specifically, the sixth step proceeds in only 26% yield, and the product must be purified by preparative HPLC. Additionally, step eight proceeds in good yield, but the process involves copious volumes of the coreactants. These difficulties in steps 6-8 of the prior art synthesis are overcome in the practice of the present invention through the use of the following synthetic alternative therefor. CsCoj, DMF The poor yield in step six of the prior art synthesis procedure, in which a tetra amine alcohol is converted to a tetra-toluenesulfonamide toluenesulfonate as shown below, is the likely result of premature formation of the O-toluenesulfonate functionality (before all of the amine groups have been converted to their corresponding sulfonamides. Such a sequence of events would potentially result in unwanted intra- or inter-molecular displacement of the reactive O-toluenesulfonate by unprotected amine groups, thereby generating numerous undesirable side- products. This problem is overcome in the aforementioned alternative synthesis scheme of the present invention by reacting the tetra-a ine alcohol with trifluoroacetic anhydride. Trifluoroacetates, being much poorer leaving groups than toluenesulfonates, are not vulnerable to analogous side reactions. In fact, the easy hydrolysis of trifluoroacetate groups, as reported in Greene and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, Inc., New York, p. 94, 1991., suggests that addition of methanol to the reaction mixture following consumption of all amines should afford the tetra-fluoroacetamide alcohol as a substantially exclusive product. Conversion of the tetra-fluoroacetamide alcohol to the corresponding toluenesulfonate provides a material which is expected to cyclize analogously to the tetra-toluenesulfonamide toluenesulfonate of the prior art. The cyclic tetra- amide product of the cyclization of the toluenesulfonate of tetra-fluoroacetamide alcohol, in methanolic sodium hydroxide at 15-25 °C for 1 hour, should afford nitro-benzyl-DOTA as a substantially exclusive product. As a result, the use of trifluoracetamide protecting groups circumvents the difficulties associated with cleavage of the very stable toluenesulfonamide protecting group, which involves heating with a large excess of sulfuric acid followed by neutralization with copious volumes of barium hydroxide. Alternatively, the problems in the prior art with respect to conversion of ll-(p-nitrobenzyl) - N,N' ,N" ,N'" ,0-pentakis(tosyl-sulfonyl)-3,6,9,12- tetraazadodecanol to nitro-benzyl-DOTA may be overcome by the following procedure: transient protection of the hydroxy group; tosylation of the free amines; deprotection of the transiently protected hydroxy group; tosylation of the deprotected hydroxy group; and an intramolecular tosylate cyclization reaction to form a twelve-membered DOTA ring. This procedure is analogous to the one described in Example XVII with respect to the intermediate 2- (p-nitrobenzyl) - N,N',N",N'" ,0-pentakis(tosyl-sulfonyl)-3,6,9,12- tetraazadodecanol. Another alternative route to nitro-benzyl-DOTA is shown below. This alternative procedure involves the cyclizaton of p-nitrophenylalanyltriglycine using a coupling agent, such as diethylycyanophosphate, to give the cyclic tetraamide. Subsequent borane reduction provides 2- (p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane, a common precursor used in published routes to DOTA including the Renn and Meares article referenced above. This alternative procedure of the present invention offers a synthetic pathway that is considerably shorter than the prior art Renn and Meares route, requiring two rather than four steps between p-nitrophenylalanyltriglycine to the tetraamine. The procedure of the present invention also avoids the use of tosyl amino protecting groups, which were prepared in low yield and required stringent conditions for removal. Also, the procedure of the present invention poses advantages over the route published by Gansow et al., U.S. Patent No. 4,923,985, because the crucial cyclization step is intramolecular rather than intermolecular. Intramolecular reactions typically proceed in higher yield and do not require high dilution techniques necessary for successful intermolecular reactions. Further alternative routes to nitro-benzyl-DOTA include the triamide route of Figure 12, the direct peptide cyclization routes of Figures 13 and 14 and the reverse direct peptide cyclization route of Figure 15, all of which are described in more detail in Example XV. Additional routes to DOTA chelate compounds are set forth in Example XVII. These routes include the production of DOTA chelate compounds employing a phenylalanine or substituted phenylalanine starting material. Preferable substituted phenylalanine starting materials are substituted at the para position and include N0 2 and NHR' wherein R' is an amino protecting group. Another preferred para substituent is NH-S0 2 -2,3, 6-methyl-4-methoxyphenyl. Preferred amino protecting groups include toluenesulfonyl, isonicotinyl-carbamate and 9- fluorenylmethyl carbamate. Radiolysis of Y-90 plays a role in the stability of Y-90-labeled compounds. During radiolysis, Y-90, a high energy beta-emitter that gives off a beta particle having a maximum energy of about 2.27 MeV, generates a greater number of oxidizing oxy-radicals (e.g.. superoxide anion, hydroperoxy radical, hydrogen peroxide, and the like) per particle emitted. Consequently, these active oxidants attack the Y-90- chelate complex or other constituents of the complex resulting in inter- or intra-molecular decomposition. This phenomena has been observed with respect to strong beta-emitters, such as Cu-67, 1-131, Re-186 and Re-188. The DOTA-containing compounds discussed above incorporate a N 4 macrocyclic ring structure. Studies discussed further below in Example XVIII have shown that a radioprotectant serves to stabilize Y-90- labeled DOTA-biotin compounds with respect to radiolysis. Preferred radioprotectants of the present invention are anti-oxidants (e.g. , either reduce the number or the activity of oxidizing radicals) . Exemplary radioprotectants that may be employed in the practice of the present invention are gentisic acid, ascorbic acid, nicotinic acid, ascorbyl palmitate, HOP(:0)H 2 , monothioglycerol, sodium formaldehyde sulfoxylate, Na 2 S 2 0 5 , Na 2 S 2 0 3 , ascorbate, S0 2 , or a reducing agent combined with BHA, BHT, Pr gallate or tocopherol and the like. Ascorbic acid is a preferred radioprotectant for use in the practice of the present invention. The present invention also provides radiolabeled ligand molecules bearing chemically modified ligand components. When biotin-N-methyl-glycine-aminobenzyl- DOTA is oxidized with sodium periodate and the resultant oxidized product is labeled with Y-90, this compound exhibits the same HPLC retention time as the radiolysis product of the radiolabeled biotin-DOTA conjugate. NMR characterization of the oxidized biotin-N-methyl-glycine-aminobenzyl-DOTA is consistent with biotin-sulfoxide formation. In view of these results, it appears that radiolysis causes oxidation of the biotin heterocycle. The radiolysis and oxidized biotin products retain avidin binding capability when tested in vitro. See, for example, Example XVIII. Consequently, oxidized biotin- containing conjugates may be employed in the practice of pretargeting embodiments of the present invention. This data also indicates that radiolysis of Y-90- radiolabeled biotin does not result in the release of free Y-90. The present invention also provides an article of manufacture which includes packaging material and an active agent-ligand or active agent-anti-ligand conjugate, such as an Y-90-DOTA-biotin conjugate, contained within the packaging material, wherein the conjugate, upon administration to a mammalian recipient, is capable of localizing at a target site at which a previously administered complementary ligand/anti-ligand pair member has localized, and wherein the packaging material includes a label that identifies the ligand or anti-ligand component of the conjugate, identifies the active agent component of the conjugate and indicates an appropriate use of the conjugate in human recipients. The packaging material indicates whether the conjugate is limited to investigational use or identifies an indication for which the conjugate has been approved by the U.S. Food and Drug Administration or other similar regulatory body for use in humans. The packaging material may also include additional information including the amount of conjugate, the medium or environment in which the conjugate is dispersed, if any, lot number or other identifier, storage instructions, usage instructions, a warning with respect to any restriction upon use of the conjugate, the name and address of the company preparing and/or packaging the conjugate, and other information concerning the conjugate. The active agent-ligand conjugate or active agent- anti-ligand conjugate is preferably contained within a vial which allows the conjugate to be transported prior to use. Such conjugate is preferably vialed in a sterile, pyrogen-free environment. Alternatively, the conjugate may be lyophilized prior to packaging. In this circumstance, instructions for preparing the lyophilized conjugate for administration to a recipient may be included on the label. One component to be administered in a preferred two-step pretargeting protocol is a targeting moiety- anti-ligand or a targeting moiety-ligand conjugate. In three-step pretargeting, a preferred component for administration is a targeting moiety-ligand conjugate. A preferred targeting moiety useful in these embodiments of the present invention is a monoclonal antibody. Protein-protein conjugations are generally problematic due to the formation of undesirable byproducts, including high molecular weight and cross- linked species, however. A non-covalent synthesis technique involving reaction of biotinylated antibody with streptavidin has been reported to result in substantial byproduct formation. Also, at least one of the four biotin binding sites on the streptavidin is used to link the antibody and streptavidin, while another such binding site may be sterically unavailable for biotin binding due to the configuration of the εtreptavidin-antibody conjugate. Thus, covalent streptavidin-antibody conjugation is preferred, but high molecular weight byproducts are often obtained. The degree of crosslinking and aggregate formation is dependent upon several factors, including the level of protein derivitization using heterobifunctional crosslinking reagents. Sheldon et al., Appl. Radiat. Isot. 43: 1399-1402, 1992, discuss preparation of covalent thioether conjugates by reacting succinimidyl 4-(N-maleimidomethyl)cyclohexane -1-carboxylate (SMCC) -derivitized antibody and iminothiolane-derivitized streptavidin. Streptavidin-proteinaceous targeting moiety conjugates are preferably prepared as described in Example XI below, with the preparation involving the steps of: preparation of SMCC-derivitized streptavidin; preparation of DTT-reduced proteinaceous targeting moiety; conjugation of the two prepared moieties; and purification of the monosubstituted and/or disubstituted (with streptavidin) conjugate from crosslinked (antibody-streptavidin-antibody) and aggregate species. The purified fraction is preferably further characterized by one or more of the following techniques: HPLC size exclusion, SDS-PAGE, immunoreactivity, biotin binding capacity and in vivo studies. Alternatively, thioether conjugates useful in the practice of the present invention may be formed using other thiolating agents, such as SPDP, iminothiolane, SATA or the like, or other thio-reactive heterobifunctional cross linkers, such as m- maleimidobenzoyl-N-hydroxysuccinimide ester, N- succinimidyl(4-iodoacetyl)aminobenzoate or the like. Streptavidin-proteinaceous targeting moiety conjugates of the present invention can also be formed by conjugation of a lysine epsilon amino group of one protein with a maleimide-derivitized form of the other protein. For example, at pH 8-10, lysine epsilon amino moieties react with protein maleimides, prepared, for instance, by treatment of the protein with SMCC, to generate stable amine covalent conjugates. In addition, conjugates can be prepared by reaction of lysine epsilon amino moieties of one protein with aldehyde functionalities of the other protein. The resultant imine bond is reducible to generate the corresponding stable amine bond. Aldehyde functionalities may be generated, for example, by oxidation of protein sugar residues or by reaction with aldehyde-containing heterobifunctional cross linkers. Another method of forming streptavidin-targeting moiety conjugates involves immobilized iminobiotin that binds SMCC-derivitized streptavidin. In this conjugation/purification method, the reversible binding character of iminobiotin (immobilized) to streptavidin is exploited to readily separate conjugate from the unreacted targeting moiety. Iminobiotin binding can be reversed under conditions of lower pH and elevated ionic strength, e.g. , NH 2 0Ac, pH 4 (50 mM) with 0.5 M NaCl. For streptavidin, for example, the conjugation/ purification proceeds as follows: - SMCC-derivitized streptavidin is bound to immobilized iminobiotin (Pierce Chemical Co. , St. Louis, Missouri) , preferably in column format; - a molar excess (with respect to streptavidin) of DTT-reduced antibody (preferably free of reductant) is added to the nitrogen-purged, phosphate-buffered iminobiotin column wherein the SMCC-streptavidin is bound (DTT-reduced antibody will saturate the bound SMCC-streptavidin, and unbound reduced antibody passing through the column can be reused) ; - the column is washed free of excess antibody; and - a buffer that lowers the pH and increases ionic strength is added to the column to elute εtreptavidin- antibody conjugate in pure form. As indicated above, targeting moiety-mediated ligand-anti-ligand pretargeting involves the localization of either targeting moiety-ligand or targeting moiety-anti-ligand at target tissue. Often, peak uptake to such target tissue is achieved before the circulating level of targeting moiety-containing conjugate in the blood is sufficiently low to permit the attainment of an optimal target-to-non-target conjugate ratio. To obviate this problem, two approaches are useful. The first approach allows the targeting moiety-containing conjugate to clear from the blood by "natural" or endogenous clearance mechanisms. This method is complicated by variations in systemic clearance of proteins and by endogenous ligand or anti-ligand. For example, endogenous biotin may interfere with the preservation of biotin binding sites on a streptavidin-targeting moiety conjugate. The second approach for improving targeting moiety-ligand or targeting moiety-anti-ligand conjugate target-to-blood ratio "chases" the conjugate from the circulation through m vivo complexation of conjugate with a molecule constituting or containing the complementary anti-ligand or ligand. When biotinylated antibodies are used as a ligand-targeting moiety conjugate, for example, avidin forms relatively large aggregated species upon complexation with the circulating biotinylated antibody, which aggregated species are rapidly cleared from the blood by the RES uptake. See, for example, U.S. Patent No. 4,863,713. One problem with this method, however, is the potential for cross-linking and internalizing tumor- bound biotinylated antibody by avidin. When avidin-targeting moiety conjugates are employed, poly-biotinylated transferrin has been used to form relatively large aggregated species that are cleared by RES uptake. See, for example, Goodwin, J. Nucl. Med. 33 (10) : 1816-18. 1992) . Poly-biotinylated transferrin also has the potential for cross-linking and internalizing tumor-bound avidinylated-targeting moiety, however. In addition, both "chase" methodologies involve the prolonged presence of aggregated moieties of intermediate, rather than large, size (which are not cleared as quickly as large size particles by RES uptake) , thereby resulting in serum retention of subsequently administered ligand- active agent or anti-ligand active agent. Such serum retention unfavorably impacts the target cell-to-blood targeting ratio. The present invention provides clearing agents of protein and non-protein composition having physical properties facilitating use for in vivo complexation and blood clearance of anti-ligand/ligand (e.g.. avidin/biotin)-targeting moiety (e.g. , antibody) conjugates. These clearing agents are useful in improving the target:blood ratio of targeting moiety conjugate. Other applications of these clearing agents include lesional imaging or therapy involving blood clots and the like, employing antibody-active agent delivery modalities. For example, efficacious anti-clotting agent provides rapid target localization and high target:non-target targeting ratio. Active agents administered in pretargeting protocols of the present invention using efficient clearing agents are targeted in the desirable manner and are, therefore, useful in the imaging/therapy of conditions such as pulmonary embolism and deep vein thrombosis. Clearing agents useful in the practice of the present invention preferably exhibit one or more of the following characteristics: - rapid, efficient complexation with targeting moiety-ligand (or anti-ligand) conjugate in vivo; - rapid clearance from the blood of targeting moiety conjugate capable of binding a subsequently administered complementary anti-ligand or ligand containing molecule; - high capacity for clearing (or inactivating) large amounts of targeting moiety conjugate; and - low immunogenicity. Preferred clearing agents include hexose-based and non-hexose based moieties. Hexose-based clearing agents are molecules that have been derivatized to incorporate one or more hexoses (six carbon sugar moieties) recognized by Ashwell receptors or other receptors such as the mannose/N-acetylglucosamine receptor which are associated with endothelial cells and/or Kupffer cells of the liver or the mannose 6- phosphate receptor. Exemplary of such hexoses are galactose, mannose, mannose 6-phosphate, N- acetylglusosamine and the like. Other moieties recognized by Ashwell receptors, including glucose, N- galactosamine, N-acetylgalactosamine, thioglycosides of galactose and, generally, D-galactosides and glucosides or the like may also be used in the practice of the present invention. Galactose is the prototypical clearing agent hexose derivative for the purposes of this description. Galactose thioglycoside conjugation to a protein is preferably accomplished in accordance with the teachings of Lee et al., "2-Imino- 2-methoxyethyl 1-Thioglycosides: New Reagents for Attaching Sugars to Proteins," Biochemistry. 15(18) : 3956, 1976. Another useful galactose thioglycoside conjugation method is set forth in Drantz et al, "Attachment of Thioglycosides to Proteins: Enhancement of Liver Membrane Binding," Biochemistry. 15(18) : 3963, 1976. Thus, galactose-based and non- galactose based molecules are discussed below. Protein-type galactose-based clearing agents include proteins having endogenous exposed galactose residues or which have been derivatized to expose or incorporate such galactose residues. Exposed galactose residues direct the clearing agent to rapid clearance by endocytosis into the liver through specific receptors therefor (Ashwell receptors) . These receptors bind the clearing agent, and induce endocytosis into the hepatocyte, leading to fusion with a lysosome and recycle of the receptor back to the cell surface. This clearance mechanism is characterized by high efficiency, high capacity and rapid kinetics. An exemplary clearing agent of the protein- based/galactose-bearing variety is the asialoorosomucoid derivative of human alpha-1 acid glycoprotein (orosomucoid, molecular weight = 41,000 Dal, isoelectric point = 1.8-2.7) . The rapid clearance from the blood of asialoorosomucoid has been documented by Galli, et al., J. of Nucl. Med. Allied Sci. 32 (2) : 110-16, 1988. Treatment of orosomucoid with neuraminidase removes sialic acid residues, thereby exposing galactose residues. Other such derivatized clearing agents include, for example, galactosylated albumin, galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin, asialoceruloplasmin and the like. Human serum albumin (HSA) , for example, may be employed in a clearing agent of the present invention as follows: (Hexose) m —Human Serum Albumin (HSA)—(Ligand) n , wherein n is an integer from 1 to about 10 and m is an integer from 1 to about 25 and wherein the hexose is recognized by Ashwell receptors. In a preferred embodiment of the present invention the ligand is biotin and the hexose is galactose. More preferably, HSA is derivatized with from 10-20 galactose residues and 1-5 biotin residues. Still more preferably, HSA clearing agents of the present invention are derivatized with from about 12 to about 15 galactoses and 3 biotins. Derivatization with both galactose and biotin are conducted in a manner sufficient to produce individual clearing agent molecules with a range of biotinylation levels that averages a recited whole number, such as 1, biotin. Derivatization with 3 biotins, for example, produces a product mixture made up of individual clearing agent molecules, substantially all of which having at least one biotin residue. Derivatization with 1 biotin produces a clearing agent product mixture, wherein a significant portion of the individual molecules are not biotin derivatized. The whole numbers used in this description refer to the average biotinylation of the clearing agents under discussion. In addition, clearing agents based upon human proteins, especially human serum proteins, such as, for example, orosomucoid and human serum albumin, human IgG, human-anti-antibodies of IgG and IgM class and the like, are less immunogenic upon administration into the serum of a human recipient. Another advantage of using asialoorosomucoid is that human orosomucoid is commercially available from, for example, Sigma Chemical Co, St. Louis, Missouri. One way to prevent clearing agent compromise of target-bound conjugate through direct complexation is through use of a clearing agent of a size sufficient to render the clearing agent less capable of diffusion into the extravascular space and binding to target- associated conjugate. This strategy is useful alone or in combination with the aforementioned recognition that exposed galactose residues direct rapid liver uptake. This size-exclusion strategy enhances the effectiveness of non-galactose-based clearing agents of the present invention. The combination (exposed galactose and size) strategy improves the effectiveness of "protein-type" or "polymer-type" galactose-based clearing agents. Galactose-based clearing agents include galactosylated, biotinylated proteins (to remove circulating streptavidin-targeting moiety conjugates, for example) of intermediate molecular weight (ranging from about 40,000 to about 200,000 Dal), such as biotinylated asialoorosomucoid, galactosyl-biotinyl- human serum albumin or other galactosylated and biotinylated derivatives of non-immunogenic soluble natural proteins, as well as biotin- and galactose- derivatized polyglutamate, polylysine, polyarginine, polyaspartate and the like. High molecular weight moieties (ranging from about 200,000 to about 1,000,000 Dal) characterized by poor target access, including galactosyl-biotinyl-IgM or -IgG (approximately 150,000 Dal) molecules, as well as galactose- and biotin-derivatized transferrin conjugates of human serum albumin, IgG and IgM molecules and the like, can also be used as clearing agents of the claimed invention. Chemically modified polymers of intermediate or high molecular weight (ranging from about 40,000 to about 1,000,000 Dal), such as galactose- and biotin-derivatized dextran, hydroxypropylmethacrylamide polymers, polyvinylpyrrolidone-polystyrene copolymers, divinyl ether-maleic acid copolymers, pyran copolymers, or PEG, also have utility as clearing agents in the practice of the present invention. In addition, rapidly clearing biotinylated liposomes (high molecular weight moieties with poor target access) can be derivatized with galactose and biotin to produce clearing agents for use in the practice of the present invention. A further class of clearing agents useful in the present invention involve small molecules (ranging from about 500 to about 10,000 Dal) derivatized with galactose and biotin that are sufficiently polar to be confined to the vascular space as an in vivo volume of distribution. More specifically, these agents exhibit a highly charged structure and, as a result, are not readily distributed into the extravascular volume, because they do not readily diffuse across the lipid membranes lining the vasculature. Exemplary of such clearing agents are mono- or poly-biotin-derivatized 6,6'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo) bis[4-amino-5-hydroxy-l,3-naphthalene disulfonic acid] tetraεodium salt, mono- or poly-biotinyl-galactose- derivatized polysulfated dextran-biotin, mono- or poly-biotinyl-galactose-derivatized dextran-biotin and the like. The galactose-exposed or -derivatized clearing agents are preferably capable of (1) rapidly and efficiently complexing with the relevant ligand- or anti-ligand-containing conjugates via ligand-anti- ligand affinity; and (2) clearing such complexes from the blood via the galactose receptor, a liver specific degradation syεtem, aε oppoεed to aggregating into complexes that are taken up by the generalized RES system, including the lung and spleen. Additionally, the rapid kinetics of galactose-mediated liver uptake, coupled with the affinity of the ligand-anti-ligand interaction, allow the use of intermediate or even low molecular weight carriers. Non-galactose residue-bearing moieties of low or intermediate molecular weight (ranging from about 40,000 to about 200,000 Dal) localized in the blood may equilibrate with the extravascular space and, therefore, bind directly to target-associated conjugate, compromising target localization. In addition, aggregation-mediated clearance mechanisms operating through the RES system are accomplished using a large stoichiometric exceεε of clearing agent. In contrast, the rapid blood clearance of galactose- based clearing agents used in the present invention prevents equilibration, and the high affinity ligand- anti-ligand binding allows the use of low stoichiometric amounts of such galactose-based clearing agents. This feature further diminishes the potential for galactose-based clearing agents to compromise target-asεociated conjugate, becauεe the absolute amount of such clearing agent administered is decreased. Clearing agent evaluation experimentation involving galactose- and biotin-derivatized clearing agents of the present invention is detailed in Examples XIII and XVI. Specific clearing agents of the present invention that were examined during the Example XVI experimentation are (1) asialooroso ucoid- biotin, (2) human serum albumin derivatized with galactose and biotin, and (3) a 70,000 dalton molecular weight dextran derivatized with both biotin and galactose. The experimentation showed that proteins and polymers are derivatizable to contain both galactose and biotin and that the resultant derivatized molecule is effective in removing circulating streptavidin-protein conjugate from the serum of the recipient. Biotin loading was varied to determine the effects on both clearing the blood pool of circulating avidin-containing conjugate and the ability to deliver a subsequently administered biotinylated iεotope to a target site recognized by the streptavidin-containing conjugate. The effect of relative doses of the administered components with respect to clearing agent efficacy was also examined. Protein-type and polymer-type non-galactose-based clearing agents include the agentε described above, absent galactose exposure or derivitization and the like. Theεe clearing agentε act through an aggregation-mediated RES mechanism. In these embodiments of the present invention, the clearing agent used will be εelected on the basis of the target organ to which access of the clearing agent is to be excluded. For example, high molecular weight (ranging from about 200,000 to about 1,000,000 Dal) clearing agents will be used when tumor targets or clot targets are involved. Another class of clearing agentε includes agents that do not remove circulating ligand or anti- ligand/targeting moiety conjugates, but instead "inactivate" the circulating conjugates by blocking the relevant anti-ligand or ligand binding sites thereon. These "cap-type" clearing agents are preferably small (500 to 10,000 Dal) highly charged molecules, which exhibit physical characteristics that dictate a volume of distribution equal to that of the plasma compartment (i.e. , do not extravasate into the extravascular fluid volume) . Exemplary cap-type clearing agents are poly-biotin-derivatized 6,6'- [(3,3'-dimethyl[1,1'-biphenyl]-4, 4'-diyl) bis (azo) bis[4-amino-5-hydroxy-l, 3-naphthalene disulfonic acid] tetrasodium salt, poly-biotinyl-derivatized polysulfated dextran-biotin, mono- or poly-biotinyl- derivatized dextran-biotin and the like. Cap-type clearing agents are derivatized with the relevant anti-ligand or ligand, and then administered to a recipient of previously administered ligand/ or anti-ligand/targeting moiety conjugate. Clearing agent-conjugate binding therefore diminishes the ability of circulating conjugate to bind any subsequently administered active agent-ligand or active agent-anti-ligand conjugate. The ablation of active agent binding capacity of the circulating conjugate increases the efficiency of active agent delivery to the target, and increases the ratio of target-bound active agent to circulating active agent by preventing the coupling of long-circulating serum protein kinetics with the active agent. Alεo, confinement of the clearing agent to the plasma compartment prevents compromise of target-associated ligand or anti-ligand. Clearing agents of the present invention may be administered in εingle or multiple doses. A single dose of biotinylated clearing agent, for example, produces a rapid decrease in the level of circulating targeting moiety-streptavidin, followed by a small increase in that level, presumably caused, at least in part, by re-equilibration of targeting moiety- streptavidin within the recipient's physiological compartments. A second or additional clearing agent doεeε may then be employed to provide supplemental clearance of targeting moiety-streptavidin. Alternatively, clearing agent may be infused intravenously for a time period sufficient to clear targeting moiety-streptavidin in a continuous manner. Other types of clearing agents and clearance systems are also useful in the practice of the present invention to remove circulating targeting moiety- ligand or -anti-ligand conjugate from the recipient's circulation. Particulate-based clearing agents, for example, are diεcuεεed in Example IX. In addition, extracorporeal clearance systems are discussed in Example IX. .In vivo clearance protocols employing arterially inserted proteinaceous or polymeric multiloop devices are also described in Example IX. One embodiment of the present invention in which rapid acting clearing agents are useful is in the delivery of Auger emitters, such as 1-125, 1-123, Er- 165, Sb-119, Hg-197, Ru-97, Tl-201 and 1-125 and Br- 77, or nucleus-binding drugs to target cell nuclei. In these embodiments of the present invention, targeting moieties that localize to internalizing receptors on target cell surfaces are employed to deliver a targeting moiety-containing conjugate (i.e.. a targeting moiety-anti-ligand conjugate in the preferred two-step protocol) to the target cell population. Such internalizing receptors include EGF receptors, transferrin receptors, HER2 receptors, IL-2 receptors, other interleukinε and cluεter differentiation receptorε, εomatoεtatin receptorε, other peptide binding receptorε and the like. After the paεεage of a time period εufficient to achieve localization of the conjugate to target cells, but insufficient to induce internalization of such targeted conjugates by those cells through a receptor- mediated event, a rapidly acting clearing agent iε administered. In a preferred two-step protocol, an active agent-containing ligand or anti-ligand conjugate, such as a biotin-Auger emitter or a biotin- nucleus acting drug, is administered as soon as the clearing agent has been given an opportunity to complex with circulating targeting moiety-containing conjugate, with the time lag between clearing agent and active agent administration being lesε than about 24 hourε. In this manner, active agent is readily internalized through target cell receptor-mediated internalization. While circulating Auger emitters are thought to be non-toxic, the rapid, specific targeting afforded by the pretargeting protocols of the present invention increaseε the potential of shorter half-life Auger emitters, such as 1-123, which is available and capable of stable binding. In order to more effectively deliver a therapeutic or diagnostic dose of radiation to a target site, the radionuclide is preferably retained at the tumor cell surface. Loss of targeted radiation occurs as a consequence of metabolic degradation mediated by metabolically active target cell typeε, εuch aε tumor or liver cellε. Preferable agentε and protocolε within the preεent invention are therefore characterized by prolonged reεidence of radionuclide at the target cell εite to which the radionuclide haε localized and improved radiation absorbed dose deposition at that target cell site, with decreased targeted radioactivity losε reεulting from metabolism. Radionuclides that are particularly amenable to the practice of this aspect of the present invention are rhenium, iodine and like "non +3 charged" radiometals which exist in chemical forms that easily cross cell membranes and are not, therefore, inherently retained by cells. In contraεt, radionuclides having a +3 charge, such as In-Ill, Y- 90, Lu-177 and Ga-67, exhibit natural target cell retention as a result of their containment in high charge density chelates. Evidence exists that streptavidin is resiεtant to metabolic degradation. Conεequently, radionuclides bound directly or indirectly to streptavidin, rather than, for example, directly to the targeting moiety, are retained at target cell sites for extended periods of time. Streptavidin-aεεociated radionuclideε can be adminiεtered in pretargeting protocolε intravenouεly, intraarterially or the like or injected directly into leεions. Monovalent antibody fragment-streptavidin conjugate may be uεed to pretarget εtreptavidin, preferably in additional embodiments of the two-step aspect of the present invention. Exemplary monovalent antibody fragmentε useful in these embodiments are Fv, Fab, Fab' and the like. Monovalent antibody fragments, typically exhibiting a molecular weight ranging from about 25 kD (Fv) to about 50 kD (Fab, Fab'), are smaller than whole antibody and, therefore, are generally capable of greater target site penetration. Moreover, monovalent binding can result in less binding carrier restriction at the target surface (occurring during use of bivalent antibodieε, which bind strongly and adhere to target cell sites thereby creating a barrier to further egresε into εublayerε of target tiεεue) , thereby improving the homogeneity of targeting. In addition, smaller molecules are more rapidly cleared from a recipient, thereby decreasing the immunogenicity of the administered small molecule conjugate. A lower percentage of the administered dose of a monovalent fragment conjugate localizes to target in comparison to a whole antibody conjugate. The decreased immunogenicity may permit a greater initial dose of the monovalent fragment conjugate to be administered, however. A multivalent, with respect to ligand, moiety is preferably then administered. This moiety also has one or more radionuclides asεociated therewith. As a result, the multivalent moiety serves as both a clearing agent for circulating anti-ligand-containing conjugate (through cross-linking or aggregation of conjugate) and as a therapeutic agent when associated with target bound conjugate. In contrast to the internalization caused by cross-linking described above, crosε-linking at the tumor cell surface stabilizes the monovalent fragment-anti-ligand molecule and, therefore, enhances target retention, under appropriate conditions of antigen density at the target cell. In addition, monovalent antibody fragments generally do not internalize as do bivalent or whole antibodies. The difficulty in internalizing monovalent antibodies permits croεε-linking by a monovalent moiety εerves to stabilize the bound monovalent antibody through multipoint binding. This two-step protocol of the present invention has greater flexibility with respect to dosing, because the decreased fragment immunogenicity allows more streptavidin-containing conjugate, for example, to be administered, and the simultaneous clearance and therapeutic delivery removes the necesεity of a εeparate controlled clearing εtep. Another embodiment of the pretargeting methodologieε of the present invention involves the route of administration of the ligand- or anti-ligand- active agents. In these embodiments of the present invention, the active agent-ligand (e.g.. radiolabeled biotin) or -anti-ligand is administered intraarterially using an artery supplying tissue that contains the target. In the radiolabeled biotin example, the high extraction efficiency provided by avidin-biotin interaction facilitates delivery of very high radioactivity levels to the target cells, provided the radioactivity specific activity levels are high. The limit to the amount of radioactivity delivered therefore becomes the biotin binding capacity at the target (i.e. , the amount of antibody at the target and the avidin equivalent attached thereto) . For these embodiments of the pretargeting methods of the present invention, particle emitting therapeutic radionuclides resulting from transmutation processes (without non-radioactive carrier forms present) are preferred. Exemplary radionuclides include Y-90, Re-188, At-211, Bi-212 and the like. Other reactor-produced radionuclides are useful in the practice of these embodiments of the present invention, if they are able to bind in amounts delivering a therapeutically effective amount of radiation to the target. A therapeutically effective amount of radiation ranges from about 1500 to about 10,000 cGy depending upon several factors known to nuclear medicine practitioners. Intraarterial administration pretargeting can be applied to targets present in organs or tissues for which supply arterieε are acceεεible. Exemplary applications for intraarterial delivery aspects of the pretargeting methods of the present invention include treatment of liver tumors through hepatic artery administration, brain primary tumorε and metaεtases through carotid artery administration, lung carcinomaε through bronchial artery administration and kidney carcinomas through renal artery administration. Intraarterial administration pretargeting can be conducted using chemotherapeutic drug, toxin and anti- tumor active agents as discuεεed below. High potency drugs, lymphokines, such aε IL-2 and tumor necroεiε factor, drug/lymphokine-carrier-biotin moleculeε, biotinylated drugε/lymphokineε, and drug/lymphokine/ toxin-loaded, biotin-derivitized lipoεomeε are exemplary of active agentε and/or doεage forms useful for the delivery thereof in the practice of this embodiment of the present invention. In embodiments of the present invention employing radionuclide therapeutic agents, the rapid clearance of nontargeted therapeutic agent decreases the exposure of non-target organs, such as bone marrow, to the therapeutic agent. Consequently, higher doses of radiation can be administered absent dose limiting bone marrow toxicity. In addition, pretargeting methodε of the preεent invention optionally include administration of short duration bone marrow protecting agents, such as WR 2721. As a result, even higher doεes of radiation can be given, absent dose limiting bone marrow toxicity. An additional aspect of the present invention is directed to the use of targeting moieties that are monoclonal antibodies or fragments thereof that localize to an antigen that is recognized by the antibody NR-LU-10. Such monoclonal antibodies or fragments may be murine or of other non-human mammalian origin, chimeric, humanized or human. NR-LU-10 is a 150 kilodalton molecular weight IgG2b monoclonal antibody that recognizes an approximately 40 kilodalton glycoprotein antigen expressed on most carcinomaε. in vivo εtudieε in mice using an antibody specific for the NR-LU-10 antigen revealed that such antibody was not rapidly internalized, which would have prevented localization of the subsequently administered active-agent- containing conjugate to the target εite. NR-LU-10 iε a well characterized pancarcinoma antibody that haε been εafely adminiεtered to over 565 patientε in human clinical trialε. The hybridoma secreting NR-LU-10 waε developed by fuεing mouεe splenocyteε immunized with intact cells of a human εmall cell lung carcinoma with P3 x 63/Ag8UI murine myeloma cells. After establiεhing a seed lot, the hybridoma was grown via in vitro cell culture methods, purified and verified for purity and sterility. Radioimmunoasεayε, immunoprecipitation and Fluoreεcence-Activated Cell Sorter (FACS) analyεis were used to obtain reactivity profileε of NR-LU-10. The NR-LU-10 target antigen was present on either fixed cultured cells or in detergent extracts of various types of cancer cells. For example, the NR- LU-10 antigen iε found in εmall cell lung, non-εmall cell lung, colon, breaεt, renal, ovarian, pancreatic, and other carcinoma tiεsues. Tumor reactivity of the NR-LU-10 antibody iε set forth in Table A, while NR- LU-10 reactivity with normal tissues is set forth in Table B. The values in Table B are obtained as described below. Positive NR-LU-10 tissue reactivity indicates NR-LU-10 antigen expression by such tissues. The NR-LU-10 antigen has been further described by Varki et al., "Antigens Associated with a Human Lung Adenocarcinoma Defined by Monoclonal Antibodies," Cancer Research, 44: 681-687, 1984, and Okabe et al., "Monoclonal Antibodies to Surface Antigens of Small Cell Carcinoma of the Lung," Cancer Research, 44: 5273-5278, 1984. The tissue specimens were scored in accordance with three reactivity parameters: (1) the intensity of the reaction; (2) the uniformity of the reaction within the cell type; and (3) the percentage of cells reactive with the antibody. These three values are combined into a single weighted comparative value between 0 and 500, with 500 being the most intenεe reactivity. This comparative value facilitateε compariεon of different tissues. Table B includes a summary reactivity value, the number of tissue samples examined and the number of samples that reacted positively with NR-LU-10. Methods for preparing antibodies that bind to epitopeε of the NR-LU-10 antigen are deεcribed in U.S. Patent No. 5,084,396. Briefly, such antibodies may be prepared by the following procedure: - absorbing a first monoclonal antibody directed against a first epitope of a polyvalent antigen onto an inert, insoluble matrix capable of binding immunoglobulin, thereby forming an immunosorbent; - combining the immunosorbent with an extract containing polyvalent NR-LU-10 antigen, forming an insolubilized immune complex wherein the first epitope is masked by the first monoclonal antibody; - immunizing an animal with the insolubilized immune complex; fusing spleen cells from the immunized animal to myeloma cells to form a hybridoma capable of producing a second monoclonal antibody directed againεt a second epitope of the polyvalent antigen; culturing the hybridoma to produce the second monoclonal antibody; and collecting the second monoclonal antibody as a product of the hybridoma. Consequently, monoclonal antibodies NR-LU-01, NR-LU-02 and NR-LU-03, prepared in accordance with the procedures described in the aforementioned patent, are exemplary targeting moieties useful in this aspect of the present invention. Additional antibodies reactive with the NR-LU-10 antigen may also be prepared by standard hybridoma production and screening techniques. Any hybridoma clones so produced and identified may be further screened as described above to verify antigen and tissue reactivity. Table A TUMOR REACTIVITY OF ANTIBODY Rated from 0-3. with 3 representing highest intensity Percentage of cells stained within the examined tissue section. Rates from 0-3. with 3 representing highest uniformity Table B Table B Cont'd Table B Cont'd Table B Cont'd 69 Table Cont'd Table B Cont'd 71 Table B Cont'd 72 Table B Cont'd The invention is further described through presentation of the following examples. These examples are offered by way of illustration, and not by way of limitation. Example I Synthesis of a Chelate-Biotin Conjugate A chelating compound that contains an N 3 S chelating core was attached via an amide linkage to biotin. Radiometal labeling of an exemplary chelate-biotin conjugate is illustrated below. X = (CH 2 )o . short chain M = Tc or Rc X = CO(CH 2 )5NH, long chain R 1 » H or CH 2 C0 2 H The spacer group "X" permits the biotin portion of the conjugate to be sterically available for avidin binding. When "R 1 " is a carboxylic acid substituent (for instance, CH 2 C00H) , the conjugate exhibits improved water solubility, and further directs jln vivo excretion of the radiolabeled biotin conjugate toward renal rather than hepatobiliary clearance. Briefly, N-α-Cbz-N-Σ-t-BOC protected lysine was converted to the succinimidyl ester with NHS and DCC, and then condensed with aspartic acid β—t-butyl ester. The resultant dipeptide was activated with NHS and DCC , and then condensed with glycine t-butyl ester . The Cbz group was removed by hydrogeno lysis , and the amine was acylated using tetrahydropyranyl mercaptoacetic acid succinimidyl ester , yielding S- ( tetrahydropyranyl) - ercaptoacetyl-lysine . Trif luoroacetic acid cleavage of the N-t-BOC group and t-butyl esters , followed by condensation with LC- biotin-NHS ester provided (Σ-caproylamide biotin) - aspartyl glycine . This synthetic method is illustrated below . -E NMR: (CD 3 OD, 200 MHz Varian) : 1.25-1.95 (m, 24H) , 2.15-2.25 (broad t, 4H) , 2.65-3.05 (m, 4H) , 3.30-3.45 (dd, 2H) , 3.50-3.65 (ddd, 2H) , 3.95 (broad s, 2H) , 4.00-4.15 ( , 1H) , 4.25-4.35 (m, 1H) , 4.45-4.55 (m, 1H) , 4.7-5.05 (m overlapping with HOD) . Elemental Analysis : C , H , N for C 35 H 57 N 7 O u S 2 - H 2 0 calculated : 50 . 41 , 7 . 13 , 11 . 76 found : 50 . 13 , 7 . 14 , 11 . 40 Example II Preparation of a Technetium or Rhenium Radiolabeled Chelate-Biotin Conjugate The chelate-biotin conjugate of Example I was radiolabeled with either 99 Tc pertechnetate or 186 Re perrhenate. Briefly, 99m Tc pertechnetate was reduced with stannous chloride in the presence of sodium gluconate to form an intermediate Tc-gluconate complex. The chelate-biotin conjugate of Example I was added and heated to 100°C for 10 min at a pH of about 1.8 to about 3.3. The solution was neutralized to a pH of about 6 to about 8, and yielded an N 3 S- coordinated 99m Tc-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60% acetonitrile in 1% acetic acid demonstrated two anomers at 97% or greater radiochemical yield using δ (gamma ray) detection. Alternatively, 186 Re perrhenate was spiked with cold ammonium perrhenate, reduced with stannous chloride, and complexed with citrate. The chelate-biotin conjugate of Example I was added and heated to 90°C for 30 min at a pH of about 2 to 3. The solution was neutralized to a pH of about 6 to about 8, and yielded an N 3 S-coordinated 186 Re-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60% acetonitrile in 1% acetic acid resulted in radiochemical yields of 85- 90%. Subsequent purification over a C-18 reverse phase hydrophobic column yielded material of 99% purity. Example III in Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates Both the ""Tc- and 186 Re-chelate-biotin conjugates were evaluated in vitro. When combined with excess avidin (about 100-fold molar excess) , 100% of both radiolabeled biotin conjugates complexed with avidin. A " ∞ Tc-biotin conjugate was subjected to various chemical challenge conditions. Briefly, "Tc-chelate- biotin conjugates were combined with avidin and passed over a 5 cm size exclusion gel filtration column. The radiolabeled biotin-avidin complexes were subjected to various chemical challenges (see Table 1) , and the incubation mixtures were centrifuged through a size exclusion filter. The percent of radioactivity retained (indicating avidin-biotin-associated radiolabel) is presented in Table 1. Thus, upon chemical challenge, the radiometal remained associated with the macromolecular complex. TABLE 1 Chemical Challenge of " ∞ Tc-Chelate- Biotin-Avidin Complexes % Radioactivity Retained 1 h. 37°C 18 h. RT 99 99 97 97 92 95 99 98 97 94 In addition, each radiolabeled biotin conjugate was incubated at about 50 μg/ml with serum; upon completion of the incubation, the samples were subjected to instant thin layer chromatography (ITLC) in 80% methanol. Only 2-4% of the radioactivity remained at the origin (i.e., associated with protein) ; this percentage was unaffected by the addition of exogenous biotin. When the samples were analyzed using size exclusion H-12 FPLC with 0.2 M phosphate as mobile phase, no association of radioactivity with serum macromolecules was observed. Each radiolabeled biotin conjugate was further examined using a competitive biotin binding assay. Briefly, solutions containing varying ratios of D- biotin to radiolabeled biotin conjugate were combined with limiting avidin at a constant total biotin:avidin ratio. Avidin binding of each radiolabeled biotin conjugate was determined by ITLC, and was compared to the theoretical maximum stoichiometric binding (as determined by the HABA spectrophotometric assay of Green, Biochem. J. 94:23c-24c, 1965). No significant difference in avidin binding was observed between each radiolabeled biotin conjugate and D-biotin. Example IV In Vivo Analysis of Radiolabeled Chelate-Biotin Conjugates Administered After Antibody Pretargeting The 186 Re-chelate-biotin conjugate of Example I was studied in an animal model of a three-step antibody pretargeting protocol. Generally, this protocol involved: (i) prelocalization of biotinylated monoclonal antibody; (ii) administration of avidin for formation of a "sandwich" at the target site and for clearance of residual circulating biotinylated antibody; and (iii) administration of the 186Re-biotin conjugate for target site localization and rapid blood clearance. Ai . Preparation and Characterization of Biotinylated Antibody Biotinylated NR-LU-10 was prepared according to either of the following procedures. The first procedure involved derivitization of antibody via lysine e-amino groups. NR-LU-10 was radioiodinated at tyrosines using chloramine T and either 125 I or 131 I sodium iodide. The radioiodinated antibody (5-10 mg/ml) was then biotinylated using biotina ido caproate NHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according to the scheme below. T - n prror tein-NH 2 The impact of lysine biotinylation on antibody immunoreactivity was examined. As the molar offering of biotin: ntibody increased from 5:1 to 40:1, biotin incorporation increased as expected (measured using the HABA assay and pronase-digested product) (Table 2, below) . Percent of biotinylated antibody immunoreactivity as compared to native antibody was assessed in a limiting antigen ELISA assay. The immunoreactivity percentage dropped below 70% at a measured derivitization of 11.1:1; however, at this level of derivitization, no decrease was observed in antigen-positive cell binding (performed with LS-180 tumor cells at antigen excess) . Subsequent experiments used antibody derivitized at a biotin:antibody ratio of 10:1. TABLE 2 Effect of Lysine Biotinylation on Immunoreactivity Alternatively, NR-LU-10 was biotinylated using thiol groups generated by reduction of cystines. Derivitization of thiol groups was hypothesized to be less compromising to antibody immunoreactivity. NR-LU-10 was radioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either 125 I or 13I I sodium iodide. Radioiodinated NR-LU-10 was incubated with 25 mM dithiothreitol and purified using size exclusion chromatography. The reduced antibody (containing free thiol groups) was then reacted with a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylene diamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO (v/v) . TABLE 3 Effect of Thiol Biotinylation on Immunoreactivity Molar Measured Immunoassessment (%) Offering Derivitization (Biotins/Ab) CBiotins/Ab) ELISA Cell Binding 10:1 4.7 114 50:1 6.5 102 100 100:1 6.1 95 100 As shown in Table 3, at a 50:1 or greater biotin:antibody molar offering, only 6 biotins per antibody were incorporated. No significant impact on immunoreactivity was observed. The lysine- and thiol-derivitized biotinylated antibodies ("antibody (lysine)" and "antibody (thiol)", respectively) were compared. Molecular sizing on size exclusion FPLC demonstrated that both biotinylation protocols yielded monomolecular (monomeric) IgGs. Biotinylated antibody (lysine) had an apparent molecular weight of 160 kD, while biotinylated antibody (thiol) had an apparent molecular weight of 180 kD. Reduction of endogenous sulfhydryls (i.e.. disulfides) to thiol groups, followed by conjugation with biotin, may produce a somewhat unfolded macromolecule. If so, the antibody (thiol) may display a larger hydrodynamic radius and exhibit an apparent increase in molecular weight by chromatographic analysis. Both biotinylated antibody species exhibited 98% specific binding to immobilized avidin-agarose. Further comparison of the biotinylated antibody species was performed using non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolving gel. Biotinylated samples were either radiolabeled or unlabeled and were combined with either radiolabeled or unlabeled avidin or streptavidin. Samples were not boiled prior to SDS-PAGE analysis. The native antibody and biotinylated antibody (lysine) showed similar migrations; the biotinylated antibody (thiol) produced two species in the 50-75 kD range. These species may represent two thiol-capped species. Under these SDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kD tetramer. When 400 μg/ml radiolabeled streptavidin was combined with 50 μg/ml biotinylated antibody (analogous to "sandwiching" conditions in vivo) , both antibody species formed large molecular weight complexes. However, only the biotinylated antibody (thiol) -streptavidin complex moved from the stacking gel into the resolving gel, indicating a decreased molecular weight as compared to the biotinylated antibody (lysine) -streptavidin complex. B_;_ Blood Clearance of Biotinylated Antibody Species Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenously administered to non-tumored nude mice at a dose of 100 μg. At 24 h post- administration of radioiodinated biotinylated NR-LU- 10, mice were intravenously injected with either saline or 400 μg of avidin. With saline administration, blood clearances for both biotinylated antibody species were biphasic and similar to the clearance of native NR-LU-10 antibody. In the animals that received avidin intravenously at 24 h, the biotinylated antibody (lysine) was cleared (to a level of 5% of injected dose) within 15 min of avidin administration (avidin:biotin = 10:1). With the biotinylated antibody (thiol) , avidin administration (10:1 or 25:1) reduced the circulating antibody level to about 35% of injected dose after two hours. Residual radiolabeled antibody activity in the circulation after avidin administration was examined in vitro using immobilized biotin. This analysis revealed that 85% of the biotinylated antibody was complexed with avidin. These data suggest that the biotinylated antibody (thiol)-avidin complexes that were formed were insufficiently crosslinked to be cleared by the RES. Blood clearance and biodistribution studies of biotinylated antibody (lysine) 2 h post-avidin or post-saline administration were performed. Avidin administration significantly reduced the level of biotinylated antibody in the blood (see Figure 1) , and increased the level of biotinylated antibody in the liver and spleen. Kidney levels of biotinylated antibody were similar. Example V In Vivo Characterization of 186 Re- Chelate-Biotin Conjugates In a Three-Step Pretargeting Protocol A 186 Re-chelate-biotin conjugate of Example I (MW ∞ 1000; specific activity = 1-2 mCi/mg) was examined in a three-step pretargeting protocol in an animal model. More specifically, 18-22 g female nude mice were implanted subcutaneously with LS-180 human colon tumor xenografts, yielding 100-200 mg tumors within 10 days of implantation. NR-LU-10 antibody (MW « 150 kD) was radiolabeled with 15 I/Chloramine T and biotinylated via lysine residues (as described in Example IV.A, above) . Avidin (MW « 66 kD) was radiolabeled with 131 I/PIP-NHS (as described for radioiodination of NR-LU-10 in Example IV.A. , above) . The experimental protocol was as follows: Group 1: Time 0, inject 100 μg 125 I-labeled, biotinylated NR-LU-10 Time 24 h, inject 400 μg 131 I-labeled avidin Time 26 h, inject 60 μg 186Re-chelate- biotin conjugate Group 2: Ti e 0, inject 400 μg 131 I-labeled avidin (control) Time 2 h, inject 60 μg 186 Re-chelate- biotin conjugate Group 3: Time 0, inject 60 μg lSi 'Tte-chelate- (control) biotin conjugate The three radiolabels employed in this protocol are capable of detection in the presence of each other. It is also noteworthy that the sizes of the three elements involved are logarithmically different — antibody = 150,000; avidin s 66,000; and biotin s 1,000. Biodistribution analyses were performed at 2, 6, 24, 72 and 120 h after administration of the 186 Re- chelate-biotin conjugate. Certain preliminary studies were performed in the animal model prior to analyzing the 186 Re-chelate-biotin conjugate in a three-step pretargeting protocol. First, the effect of biotinylated antibody on blood clearance of avidin was examined. These experiments showed that the rate and extent of avidin clearance was similar in the presence or absence of biotinylated antibody. Second, the effect of biotinylated antibody and avidin on blood clearance of the 186 Re-chelate- biotin conjugate was examined; blood clearance was similar in the presence or absence of biotinylated antibody and avidin. Further, antibody immunoreactivity was found to be uncompromised by biotinylation at the level tested. Third, tumor uptake of biotinylated antibody administered at time 0 or of avidin administered at time 24 h was examined. The results of this experimentation are shown in Fig. 1. At 25 h, about 350 pmol/g biotinylated antibody was present at the tumor; at 32 h the level was about 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100 pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and 0 pmol/g, respectively. From the same experiment, tumor to blood ratios were determined for biotinylated antibody and for avidin. From 32 h to 120 h, the ratios of tumor to blood were very similar. Rapid and efficient removal of biotinylated antibody from the blood by complexation with avidin was observed. Within two hours of avidin administration, a 10-fold reduction in blood pool antibody concentration was noted (Fig. 1) , resulting in a sharp increase in tumor to blood ratios. Avidin is cleared rapidly, with greater than 90% of the injected dose cleared from the blood within 1 hour after administration. The Re-186-biotin chelate is also very rapidly cleared, with greater than 99% of the injected dose cleared from the blood by 1 hour after administration. The three-step pretargeting protocol (described for Group 1, above) was then examined. More specifically, tumor uptake of the 186 Re-chelate-biotin conjugate in the presence or absence of biotinylated antibody and avidin was determined. In the absence of biotinylated antibody and avidin, the 186 Re-chelate- biotin conjugate displayed a slight peak 2 h post- injection, which was substantially cleared from the tumor by about 5 h. In contrast, at 2 h post- injection in the presence of biotinylated antibody and avidin (specific) , the 186 Re-chelate-biotin conjugate reached a peak in tumor approximately 7 times greater than that observed in the absence of biotinylated antibody and avidin. Further, the specifically bound 186 Re-chelate-biotin conjugate was retained at the tumor at significant levels for more than 50 h. Tumor to blood ratios determined in the same experiment increased significantly over time (i.e., T:B = ~ 8 at 30 h; α 15 at 100 h; » 35 at 140 h) . Tumor uptake of the 186 Re-chelate-biotin conjugate has further been shown to be dependent on the dose of biotinylated antibody administered. At 0 μg of biotinylated antibody, about 200 pmol/g of 186 Re- chelate-biotin conjugate was present at the tumor at 2 h after administration; at 50 μg antibody, about 500 pmol/g of 186 Re-chelate-biotin conjugate; and at 100 μg antibody, about 1,300 pmol/g of 186 Re-chelate-biotin conjugate. Rhenium tumor uptake via the three-step pretargeting protocol was compared to tumor uptake of the same antibody radiolabeled through chelate covalently attached to the antibody (conventional procedure) . The results of this comparison are depicted in Figure 2. Blood clearance and tumor uptake were compared for the chelate directly labeled rhenium antibody conjugate and for the three-step pretargeted sandwich. Areas under the curves (AUC) and the ratio of AUC^ OT /AUC,,,^ were determined. For the chelate directly labeled rhenium antibody conjugate, the ratio of AUC^ ^ /AUC bIood = 24055/10235 or 2.35; for the three-step pretargeted sandwich, the ratio of KϋC tum „/KUC bi∞ _ i = 46764/6555 or 7.13. Tumor uptake results are best taken in context with radioactivity exposure to the blood compartment, which directly correlates with bone marrow exposure. Despite the fact that 100-fold more rhenium was administered to animals in the three-step protocol, the very rapid clearance of the small molecule (Re- 186-biotin) from the blood minimizes the exposure to Re-186 given in this manner. In the same matched antibody dose format, direct labeled (conventional procedure) NR-LU-10 whole antibody yielded greater exposure to rhenium than did the 100-fold higher dose given in the three-step protocol. A clear increase in the targeting ratio (tumor exposure to radioactivity:blood exposure to radioactivity— AUC t u or : AUC b i ood ) was observed for three-step pretargeting (approximately 7:1) in comparison to the direct labeled antibody approach (approximately 2.4:1). Example VI Preparation of Chelate-Biotin Conjugates Having Improved Biodistribution Properties The biodistribution of ιπ In-labeled-biotin derivatives varies greatly with structural changes in the chelate and the conjugating group. Similar structural changes may affect the biodistribution of technetium- and rheniu -biotin conjugates. Accordingly, methods for preparing technetium- and rhenium-biotin conjugates having optimal clearance from normal tissue are advantageous. A. Neutral MAMA Chelate/Conjugate A neutral MAMA chelate-biotin conjugate is prepared according to the following scheme. a) MAMA ligand BH 3 1) TsCl BOCN * . C0 2 Me NaH V7 φ c u _ ^^ CH 3 CN, Δ tsoc The resultant chelate-biotin conjugate shows superior kidney excretion. Although the net overall charge of the conjugate is neutral, the polycarboxylate nature of the molecule generates regions of hydrophilicity and hydrophobicity. By altering the number and nature of the carboxylate groups within the conjugate, excretion may be shifted from kidney to gastrointestinal routes. For instance, neutral compounds are generally cleared by the kidneys; anionic compounds are generally cleared through the GI system. B. Polylvsine Derivitization Conjugates containing polylysine may also exhibit beneficial biodistribution properties. With whole antibodies, derivitization with polylysine may skew the biodistribution of conjugate toward liver uptake. In contrast, derivitization of Fab fragments with polylysine results in lower levels of both liver and kidney uptake; blood clearance of these conjugates is similar to that of Fab covalently linked to chelate. An exemplary polylysine derivitized chelate-biotin conjugate is illustrated below. Inclusion of polylysine in radiometal-chelate-biotin conjugates is therefore useful for minimizing or eliminating RES sequestration while maintaining good liver and kidney clearance of the conjugate. For improved renal excretion properties, polylysine derivatives are preferably succinylated following biotinylation. Polylysine derivatives offer the further advantages of: (1) increasing the specific activity of the radiometal-chelate-biotin conjugate; (2) permitting control of rate and route of blood clearance by varying the molecular weight of the polylysine polymer; and (3) increasing the circulation half-life of the conjugate for optimal tumor interaction. Polylysine derivitization is accomplished by standard methodologies. Briefly, poly-L-lysine is acylated according to standard amino group acylation procedures (aqueous bicarbonate buffer, pH 8, added biotin-NHS ester, followed by chelate NHS ester) . Alternative methodology involves anhydrous conditions using nitrophenyl esters in DMSO and triethyl amine. The resultant conjugates are characterized by UV and NMR spectra. The number of biotins attached to polylysine is determined by the HABA assay. Spectrophotometric titration is used to assess the extent of amino group derivitization. The radiometal-chelate-biotin conjugate is characterized by size exclusion. C. Cleavable Linkage Through insertion of a cleavable linker between the chelate and biotin portion of a radiometal- chelate-biotin conjugate, retention of the conjugate at the tumor relative to normal tissue may be enhanced. More specifically, linkers that are cleaved by enzymes present in normal tissue but deficient or absent in tumor tissue can increase tumor retention. As an example, the kidney has high levels of y- glutamyl transferase; other normal tissues exhibit in vivo cleavage of 7-glutamyl prodrugs. In contrast, tumors are generally deficient in enzyme peptidases. The glutamyl-linked biotin conjugate depicted below is cleaved in normal tissue and retained in the tumor. D. Serine Linker With O-Polar Substituent Sugar substitution of N 3 S chelates renders such chelates water soluble. Sulfonates, which are fully ionized at physiological pH, improve water solubility of the chelate-biotin conjugate depicted below. or SO 2 0H X = (CH 2 ) 0 or CO(CH 2 ) 4 This compound is synthesized according to the standard reaction procedures. Briefly, biocytin is condensed with N-t-BOC-(O-sulfonate or O-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose) serine biocytinamide. Subsequent cleavage of the N-t- BOC group with TFA and condensation with ligand NHS ester in DMF with triethylamine provides ligand- a idoserine(O-sulfonate or O-glucose)biocytinamide. Example VII Preparation and Characterization of PIP- Radioiodinated Biotin Radioiodinated biotin derivatives prepared by exposure of poly-L-lysine to excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimide esters in DMSO has been reported. After purification, this product was radiolabeled by the iodogen method (see, for instance, Del Rosario et al., J. Nucl. Med. 32:5, 1991, 993 (abstr.)) . Because of the high molecular weight of the resultant radioiodinated biotin derivative, only limited characterization of product (i.e., radio-HPLC and binding to immobilized streptavidin) was possible. Preparation of radioiodinated biotin according to the present invention provides certain advantages. First, the radioiodobiotin derivative is a low molecular weight compound that is amenable to complete chemical characterization. Second, the disclosed methods for preparation involve a single step and eliminate the need for a purification step. Briefly, iodobenzamide derivatives corresponding to biocytin (R = COOH) and biotinamidopentylamine (R = H) were prepared according to the following scheme. In this scheme, "X" may be any radiohalogen, including 12S I, 131 I, 123 I, 211 At and the like. Preparation of 1 was generally according to Wilbur et al., J. Nucl. Med. 3Jl:216-26, 1989, using a tributyltin intermediate. Water soluble carbodiimide was used in the above-depicted reaction, since the NHS ester 1 formed intractable mixtures with DCU. The NHS ester was not compatible with chromatography; it was insoluble in organic and aqueous solvents and did not react with biocytin in DMF or in buffered aqueous acetonitrile. The reaction between 1 and biocytin or 5-(biotinamido) pentylamine was sensitive to base. When the reaction of 1 and biocytin or the pentylamine was performed in the presence of triethyla ine in hot DMSO, formation of more than one biotinylated product resulted. In contrast, the reaction was extremely clean and complete when a suspension of 1 and biocytin (4 mg/ml) or the pentylamine (4 mg/ml) was heated in DMSO at 117°C for about 5 to about 10 min. The resultant 125 I-biotin derivatives were obtained in 94% radiochemical yield. Optionally, the radioiodinated products may be purified using C-18 HPLC and a reverse phase hydrophobic column. Hereinafter, the resultant radioiodinated products 2 . are referred to as PIP- biocytin (R = COOH) and PIP-pentylamine (R = H) . Both iodobiotin derivatives 2 exhibited >95% binding to immobilized avidin. Incubation of the products 2 . with mouse serum resulted in no loss of the ability of 2. to bind to immobilized avidin. Biodistribution studies of 2 . in male BALB/c mice showed rapid clearance from the blood (similar to 186 Re- chelate-biotin conjugates described above) . The radioiodobiotin 2 . had decreased hepatobiliary excretion as compared to the 186 Re-chelate-biotin conjugate; urinary excretion was increased as compared to the 186 Re-chelate-biotin conjugate. Analysis of urinary metabolites of 2 . indicated deiodination and cleavage of the biotin amide bond; the metabolites showed no binding to immobilized avidin. In contrast, metabolites of the 186 Re-chelate-biotin conjugate appear to be excreted in urine as intact biotin conjugates. Intestinal uptake of 2 . is <50% that of the 186 Re- chelate-biotin conjugate. These biodistribution properties of 2 . provided enhanced whole body clearance of radioisotope and indicate the advantageous use of 2 . within pretargeting protocols. 131 I-PIP-biocytin was evaluated in a two-step pretargeting procedure in tumor-bearing mice. Briefly, female nude mice were injected subcutaneously with LS-180 tumor cells; after 7 d, the mice displayed 50-100 mg tumor xenografts. At t = 0, the mice were injected with 200 μg of NR-LU-10-avidin conjugate labeled with I25 I using PIP-NHS (see Example IV.A.). At t = 36 h, the mice received 42 μg of 131 I-PIP-biocytin. The data showed immediate, specific tumor localization, corresponding to « 1.5 131 I-PIP-biocytin molecules per avidin molecule. The described radiohalogenated biotin compounds are amenable to the same types of modifications described in Example VI above for 186 Re-chelate-biotin conjugates. In particular, the following PIP- polylysine-biotin molecule is made by trace labeling polylysine with 15 I-PIP, followed by extensive biotinylation of the polylysine. Assessment of 125 I binding to immobilized avidin ensures that all radioiodinated species also contain at least an equivalent of biotin. Example VIII Preparation of Biotinylated Antibody (Thiol) Through Endogenous Antibody Sulfhydryl Groups Or Sulfhydryl-Generating Compounds Certain antibodies have available for reaction endogenous sulfhydryl groups. If the antibody to be biotinylated contains endogenous sulfhydryl groups, such antibody is reacted with N-iodoacetyl-n'-biotinyl hexylene diamine (as described in Example IV.A. , above) . The availability of one or more endogenous sulfhydryl groups obviates the need to expose the antibody to a reducing agent, such as DTT, which can have other detrimental effects on the biotinylated antibody. Alternatively, one or more sulfhydryl groups are attached to a targeting moiety through the use of chemical compounds or linkers that contain a terminal sulfhydryl group. An exemplary compound for this purpose is iminothiolane. As with endogenous sulfhydryl groups (discussed above) , the detrimental effects of reducing agents on antibody are thereby avoided. Example IX Two-Step Pretargeting Methodology That Does Not Induce Internalization A NR-LU-13-avidin conjugate is prepared as follows. Initially, avidin is derivitized with N- succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) . SMCC-derived avidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5 for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed from the mixture using preparative size exclusion HPLC. Two conjugates are obtained as products — the desired 1:1 NR-LU-13-avidin conjugate as the major product; and an incompletely characterized component as the minor product. A " ∞ Tc-chelate-biotin conjugate is prepared as in Example II, above. The NR-LU-13-avidin conjugate is administered to a recipient and allowed to clear from the circulation. One of ordinary skill in the art of radioimmunoscintigraphy is readily able to determine the optimal time for NR-LU-13-avidin conjugate tumor localization and clearance from the circulation. At such time, the """Tc-chelate-biotin conjugate is administered to the recipient. Because the ""Tc- chelate-biotin conjugate has a molecular weight of ~ 1,000, crosslinking of NR-LU-13-avidin molecules on the surface of the tumor cells is dramatically reduced or eliminated. As a result, the """Tc diagnostic agent is retained at the tumor cell surface for an extended period of time. Accordingly, detection of the diagnostic agent by imaging techniques is optimized; further, a lower dose of radioisotope provides an image comparable to that resulting from the typical three-step pretargeting protocol. Optionally, clearance of NR-LU-13-avidin from the circulation may be accelerated by plasmapheresis in combination with a biotin affinity column. Through use of such column, circulating NR-LU-13-avidin will be retained extracorporeally, and the recipient's immune system exposure to a large, proteinaceous immunogen (i.e., avidin) is minimized. Exemplary methodology for plasmapheresis/column purification useful in the practice of the present invention is discussed in the context of reducing radiolabeled antibody titer in imaging and in treating tumor target sites in U.S. Patent 5,078,673. Briefly, for the purposes of the present invention, an example of an extracorporeal clearance methodology may include the following steps: administering a ligand- or anti-ligand-targeting moiety conjugate to a recipient; after a time sufficient for localization of the administered conjugate to the target site, withdrawing blood from the recipient by, for example, plasmapheresis; separating cellular element from said blood to produce a serum fraction and returning the cellular elements to the recipient; and reducing the titer of the administered conjugate in the serum fraction to produce purified serum; infusing the purified serum back into the recipient. Clearance of NR-LU-13-avidin is also facilitated by administration of a particulate-type clearing agent (e.g. , a polymeric particle having a plurality of biotin molecules bound thereto) . Such a particulate clearing agent preferably constitutes a biodegradable polymeric carrier having a plurality of biotin molecules bound thereto. Particulate clearing agents of the present invention exhibit the capability of binding to circulating administered conjugate and removing that conjugate from the recipient. Particulate clearing agents of this aspect of the present invention may be of any configuration suitable for this purpose. Preferred particulate clearing agents exhibit one or more of the following characteristics: - microparticulate (e.g., from about 0.5 micrometers to about 100 micrometers in diameter, with from about 0.5 to about 2 micrometers more preferred), free flowing powder structure; - biodegradable structure designed to biodegrade over a period of time between from about 3 to about 180 days, with from about 10 to about 21 days more preferred, or non-biodegradable structure; - biocompatible with the recipients physiology over the course of distribution, metabolism and excretion of the clearing agent, more preferably including biocompatible biodegradation products; - and capability to bind with one or more circulating conjugates to facilitate the elimination or removal thereof from the recipient through one or more binding moieties (preferably, the complementary member of the ligand/anti-ligand pair) . The total molar binding capacity of the particulate clearing agents depends upon the particle size selected and the ligand or anti-ligand substitution ratio. The binding moieties are capable of coupling to the surface structure of the particulate dosage form through covalent or non-covalent modalities as set forth herein to provide accessible ligand or anti-ligand for binding to its previously administered circulating binding pair member. Preferable particulate clearing agents of the present invention are biodegradable or non- biodegradable microparticulates. More preferably, the particulate clearing agents are formed of a polymer containing matrix that biodegrades by random, nonenzymatic, hydrolytic scissioning. Polymers derived from the condensation of alpha hydroxycarboxylic acids and related lactones are more preferred for use in the present invention. A particularly preferred moiety is formed of a mixture of thermoplastic polyesters (e.g., polylactide or polyglycolide) or a copolymer of lactide and glycolide components, such as poly(lactide-co-glycolide) . An exemplary structure, a random poly(DL-lactide-co- glycolide) , is shown below, with the values of x and y being manipulable by a practitioner in the art to achieve desirable microparticulate properties. O O O O II ιι II ιι H- -O-CH-C-O-CH-C- -0-CH 2 -C -0-CH 2 -C - OH CH 3 CH 3 Other agents suitable for forming particulate clearing agents of the present invention include polyorthoesters and polyacetals (Polymer Letters. .18 . :293, 1980) and polyorthocarbonates (U.S. Patent No, 4,093,709) and the like. Preferred lactic acid/glycolic acid polymer containing matrix particulates of the present invention are prepared by emulsion-based processes, that constitute modified solvent extraction processes such as those described by Cowsar et al., "Poly(Lactide-Co-Glycolide) Microcapsules for Controlled Release of Steroids," Methods Enzymology, 112:101-116, 1985 (steroid entrapment in microparticulates) ; Eldridge et al., "Biodegradable and Biocompatible Poly(DL-Lactide-Co-Glycolide) Microspheres as an Adjuvant for Staphylococcal Enterotoxin B Toxoid Which Enhances the Level of Toxin-Neutralizing Antibodies," Infection and Immunity. J59 . :2978-2986, 1991 (toxoid entrapment); Cohen et al., "Controlled Delivery Systems for Proteins Based on Poly(Lactic/Glycolic Acid) Microspheres," Pharmaceutical Research, 8 (6) :713-720, 1991 (enzyme entrapment); and Sanders et al., "Controlled Release of a Luteinizing Hormone-Releasing Hormone Analogue from Poly(D,L-Lactide-Co-Glycolide) Microspheres," J. Pharmaceutical Science. 73 (9 ) : 1294- 1297, 1984 (peptide entrapment). In general, the procedure for forming particulate clearing agents of the present invention involves dissolving the polymer in a halogenated hydrocarbon solvent and adding an additional agent that acts as a solvent for the halogenated hydrocarbon solvent but not for the polymer. The polymer precipitates out from the polymer-halogenated hydrocarbon solution. Following particulate formation, they are washed and hardened with an organic solvent. Water washing and aqueous non-ionic surfactant washing steps follow, prior to drying at room temperature under vacuum. For biocompatibility purposes, particulate clearing agents are sterilized prior to packaging, storage or administration. Sterilization may be conducted in any convenient manner therefor. For example, the particulates can be irradiated with gamma radiation, provided that exposure to such radiation does not adversely impact the structure or function of the binding moiety attached thereto. If the binding moiety is so adversely impacted, the particulate clearing agents can be produced under sterile conditions. The preferred lactide/glycolide structure is biocompatible with the mammalian physiological environment. Also, these preferred sustained release dosage forms have the advantage that biodegradation thereof forms lactic acid and glycolic acid, both normal metabolic products of mammals. Functional groups required for binding moiety - particulate bonding, are optionally included in the particulate structure, along with the non-degradable or biodegradable polymeric units. Functional groups that are exploitable for this purpose include those that are reactive with ligands or anti-ligands, such as carboxy1 groups, amine groups, sulfhydryl groups and the like. Preferred binding enhancement moieties include the terminal carboxyl groups of the preferred (lactide-glycolide) polymer containing matrix or the like. A practitioner in the art is capable of selecting appropriate functional groups and monitoring conjugation reactions involving those functional groups. Advantages garnered through the use of particulate clearing agents of the type described above are as follows: - particles in the "micron" size range localize in the RES and liver, with galactose derivatization or charge modification enhancement methods for this capability available, and, preferably, are designed to remain in circulation for a time sufficient to perform the clearance function; - the size of the particulates facilitates central vascular compartment retention thereof, substantially precluding equilibration into the peripheral or extravascular compartment; - desired substituents for ligand or anti-ligand binding to the particulates can be introduced into the polymeric structure; - ligand- or anti-ligand-particulate linkages having desired properties (e.g. , serum biotinidase resistance thereby reducing the release of biotin metabolite from a particle-biotin clearing agent) and - multiple ligands or anti-ligands can be bound to the particles to achieve optimal cross-linking of circulating targeting agent-ligand or -anti-ligand conjugate and efficient clearance of cross-linked species. This advantage is best achieved when care is taken to prevent particulate aggregation both in storage and upon m. vivo administration. Clearance of NR-LU-13-avidin may also be accelerated by an arterially inserted proteinaceous or polymeric multiloop device. A catheter-like device, consisting of thin loops of synthetic polymer or protein fibers derivitized with biotin, is inserted into a major artery (e.g. , femoral artery) to capture NR-LU-13-avidin. Since the total blood volume passes through a major artery every 70 seconds, the in situ clearing device is effective to reduce circulating NR- LU-13-avidin within a short period of time. This device offers the advantages that NR-LU-13-avidin is not processed through the RES; removal of NR-LU-13- avidin is controllable and measurable; and fresh devices with undiminished binding capacity are insertable as necessary. This methodology is also useful with intraarterial administration embodiments of the present invention. An alternative procedure for clearing NR-LU-13- avidin from the circulation without induction of internalization involves administration of biotinylated, high molecular weight molecules, such as liposomes, IgM and other molecules that are size excluded from ready permeability to tumor sites. When such biotinylated, high molecular weight molecules aggregate with NR-LU-13-avidin, the aggregated complexes are readily cleared from the circulation via the RES. Example X Enhancement of Therapeutic Agent Internalization Through Avidin Crosslinking The ability of multivalent avidin to crosslink two or more biotin molecules (or chelate-biotin conjugates) is advantageously used to improve delivery of therapeutic agents. More specifically, avidin crosslinking induces internalization of crosslinked complexes at the target cell surface. Biotinylated NR-CO-04 (lysine) is prepared according to the methods described in Example IV.A. , above. Doxorubicin-avidin conjugates are prepared by standard conjugation chemistry. The biotinylated NR-CO-04 is administered to a recipient and allowed to clear from the circulation. One of ordinary skill in the art of radioimmunotherapy is readily able to determine the optimal time for biotinylated NR-CO-04 tumor localization and clearance from the circulation. At such time, the doxorubicin-avidin conjugate is administered to the recipient. The avidin portion of the doxorubicin-avidin conjugate crosslinks the biotinylated NR-CO-04 on the cell surface, inducing internalization of the complex. Thus, doxorubicin is more efficiently delivered to the target cell. In a first alternative protocol, a standard three- step pretargeting methodology is used to enhance intracellular delivery of a drug to a tumor target cell. By analogy to the description above, biotinylated NR-LU-05 is administered, followed by avidin (for blood clearance and to form the middle layer of the sandwich at the target cell-bound biotinylated antibody) . Shortly thereafter, and prior to internalization of the biotinylated NR-LU-05-avidin complex, a methotrexate-biotin conjugate is administered. In a second alternative protocol, biotinylated NR- LU-05 is further covalently linked to methotrexate. Subsequent administration of avidin induces internalization of the complex and enhances intracellular delivery of drug to the tumor target cell. In a third alternative protocol, NR-CO-04-avidin is administered to a recipient and allowed to clear from the circulation and localize at the target site. Thereafter, a polybiotinylated species (such as biotinylated poly-L-lysine, as in Example IV.B., above) is administered. In this protocol, the drug to be delivered may be covalently attached to either the antibody-avidin component or to the polybiotinylated species. The polybiotinylated species induces internalization of the (drug) -antibody-avidin- polybiotin-(drug) complex. Example XI Targeting Moiety-Anti-Ligand Conjugate for Two-Step Pretargeting In Vivo A. Preparation of SMCC-derivitized streptavidin. 31 mg (0.48 μmol) streptavidin was dissolved in 9.0 ml PBS to prepare a final solution at 3.5 mg/ml. The pH of the solution was adjusted to 8.5 by addition of 0.9 ml of 0.5 M borate buffer, pH 8.5. A DMSO solution of SMCC (3.5 mg/ml) was prepared, and 477 μl (4.8 μmol) of this solution was added dropwise to the vortexing protein solution. After 30 minutes of stirring, the solution was purified by G-25 (PD-10, Pharmacia, Piscataway, New Jersey) column chromatography to remove unreacted or hydrolyzed SMCC. The purified SMCC-derivitized streptavidin was isolated (28 mg, 1.67 mg/ml). B. Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-10 (0.42 μmol) in 15.0 ml PBS was added 1.5 ml of 0.5 M borate buffer, pH 8.5. A DTT solution, at 400 mg/ml (165 μl) was added to the protein solution. After stirring at room temperature for 30 minutes, the reduced antibody was purified by G-25 size exclusion chromatography. Purified DTT-reduced NR-LU-10 was obtained (74 mg, 2.17 mg/ml). C. Conjugation of SMCC-streptavidin to DTT- reduced NR-LU-10. DTT-reduced NR-LU-10 (63 mg, 29 ml, 0.42 μmol) was diluted with 44.5 ml PBS. The solution of SMCC-streptavidin (28 mg, 17 ml, 0.42 μmol) was added rapidly to the stirring solution of NR-LU-10. Total protein concentration in the reaction mixture was 1.0 mg/ml. The progress of the reaction was monitored by HPLC (Zorbax® GF-250, available from MacMod) . After approximately 45 minutes, the reaction was quenched by adding solid sodium tetrathionate to a final concentration of 5 mM. D. Purification of conjugate. For small scale reactions, monosubstituted and/or disubstituted (with streptavidin) conjugate was obtained using HPLC Zorbax (preparative) size exclusion chromatography. The desired monosubstituted and/or disubstituted conjugate product eluted at 14.0-14.5 min (3.0 ml/min flow rate) , while unreacted NR-LU-10 eluted at 14.5-15 min and unreacted derivitized streptavidin eluted at 19-20 min. For larger scale conjugation reactions, monosubstituted and/or disubstituted adduct is isolatable using DEAE ion exchange chromatography. After concentration of the crude conjugate mixture, free streptavidin was removed therefrom by eluting the column with 2.5% xylitol in sodium borate buffer, pH 8.6. The bound unreacted antibody and desired conjugate were then sequentially eluted from the column using an increasing salt gradient in 20 mM diethanolamine adjusted to pH 8.6 with sodium hydroxide. E. Characterization of Conjugate. 1. HPLC size exclusion was conducted as described above with respect to small scale purification. 2. SDS-PAGE analysis was performed using 5% polyacrylamide gels under non-denaturing conditions. Conjugates to be evaluated were not boiled in sample buffer containing SDS to avoid dissociation of streptavidin into its 15 kD subunits. Two product bands were observed on the gel, which correspond to the mono- and di- substituted conjugates. 3. Immunoreactivity was assessed, for example, by competitive binding ELISA as compared to free antibody. Values obtained were within 10% of those for the free antibody. 4. Biotin binding capacity was assessed, for example, by titrating a known quantity of conjugate with p-[I-125]iodobenzoylbiocytin. Saturation of the biotin binding sites was observed upon addition of 4 equivalences of the labeled biocytin. 5. rn vivo studies are useful to characterize the reaction product, which studies include, for example, serum clearance profiles, ability of the conjugate to target antigen-positive tumors, tumor retention of the conjugate over time and the ability of a biotinylated molecule to bind streptavidin conjugate at the tumor. These data facilitate determination that the synthesis resulted in the formation of a 1:1 streptavidin-NR-LU- 10 whole antibody conjugate that exhibits blood clearance properties similar to native NR-LU-10 whole antibody, and tumor uptake and retention properties at least equal to native NR-LU-10. For example. Figure 3 depicts the tumor uptake profile of the NR-LU-10-streptavidin conjugate (LU-10- StrAv) in comparison to a control profile of native NR-LU-10 whole antibody. LU-10-StrAv was radiolabeled on the streptavidin component only, giving a clear indication that LU-10-StrAv localizes to target cells as efficiently as NR-LU-10 whole antibody itself. Example XII Two-Step Pretargeting Jn Vivo A 186 Re-chelate-biotin conjugate (Re-BT) of Example I (MW « 1000; specific activity = 1-2 mCi/mg) and a biotin-iodine-131 small molecule, PIP-Biocytin (PIP- BT, MW approximately equal to 602; specific activity = 0.5-1.0 mCi/mg) , as discussed in Example VII above, were examined in a three-step pretargeting protocol in an animal model, as described in Example V above. Like Re-BT, PIP-BT has the ability to bind well to avidin and is rapidly cleared from the blood, with a serum half-life of about 5 minutes. Equivalent results were observed for both molecules in the two- step pretargeting experiments described herein. NR-LU-10 antibody (MW « 150 kD) was conjugated to streptavidin (MW « 66 kD) (as described in Example XI above) and radiolabeled with 125 I/PIP-NHS (as described for radioiodination of NR-LU-10 in Example IV.A. , above) . The experimental protocol was as follows: Time 0 inject (i.v.) 200 μg NR-LU-10-StrAv conjugate; Time 24-48 h inject (i.v.) 60-70 fold molar excess of radiolabeled biotinyl molecule ; and perform biodistributions at 2 , 6, 24, 72, 120 hours after injection of radiolabeled biotinyl molecule NR-LU-10-streptavidin has shown very consistent patterns of blood clearance and tumor uptake in the LS-180 animal model. A representative profile is shown in Figure 4. When either PIP-BT or Re-BT is administered after allowing the LU-10-StrAv conjugate to localize to target cell sites for at least 24 hours, the tumor uptake of therapeutic radionuclide is high in both absolute amount and rapidity. For PIP-BT administered at 37 hours following LU-10-StrAv (1-125) administration, tumor uptake was above 500 pMOL/G at the 40 hour time point and peaked at about 700 pMOL/G at 45 hours post-LU-10-StrAv administration. This almost instantaneous uptake of a small molecule therapeutic into tumor in stoichiometric amounts comparable to the antibody targeting moiety facilitates utilization of the therapeutic radionuclide at its highest specific activity. Also, the rapid clearance of radionuclide that is not bound to LU-10-StrAv conjugate permits an increased targeting ratio (tumor:blood) by eliminating the slow tumor accretion phase observed with directly labeled antibody conjugates. The pattern of radionuclide tumor retention is that of whole antibody, which is very persistent. Experimentation using the two-step pretargeting approach and progressively lower molar doses of radiolabeled biotinyl molecule was also conducted. Uptake values of about 20% ID/G were achieved at no- carrier added (high specific activity) doses of radiolabeled biotinyl molecules. At less than saturating doses, circulating LU-10-StrAv was observed to bind significant amounts of administered radiolabeled biotinyl molecule in the blood compartment. Example XIII Asialoorosomucoid Clearing Agent and Two-Step Pretargeting In order to maximize the targeting ratio (tumor:blood) , clearing agents were sought that are capable of clearing the blood pool of targeting moiety-anti-ligand conjugate (e.g. , LU-10-StrAv) , without compromising the ligand binding capacity thereof at the target sites. One such agent, biotinylated asialoorosomucoid, which employs the avidin-biotin interaction to conjugate to circulating LU-10-StrAv, was tested. A. Derivitization of orosomucoid. 10 mg human orosomucoid (Sigma N-9885) was dissolved in 3.5 ml of pH 5.5 0.1 M sodium acetate buffer containing 160 mM NaCl. 70 μl of a 2% (w/v) CaCl solution in deionized (D.I.) water was added and 11 μl of neuraminidase (Sigma N-7885) , 4.6 U/ml, was added. The mixture was incubated at 37°C for 2 hours, and the entire sample was exchanged over a Centricon-10® ultrafiltration device (available from Amicon, Danvers, Massachusetts) with 2 volumes of PBS. The asialoorosomucoid and orosomucoid starting material were radiolabeled with 1-125 using PIP technology, as described in Example IV above. The two radiolabeled preparations were injected i.v. into female BALB/c mice (20-25 g) , and blood clearance was assessed by serial retro-orbital eye bleeding of each group of three mice at 5, 10, 15 and 30 minutes, as well as at 1, 2 and 4 hours post- administration. The results of this experiment are shown in Figure 5, with asialoorosomucoid clearing more rapidly than its orosomucoid counterpart. In addition, two animals receiving each compound were sacrificed at 5 minutes post-administration and limited biodistributions were performed. These results are shown in Figure 6. The most striking aspects of these data are the differences in blood levels (78% for orosomucoid and 0.4% for asialoorosomucoid) and the specificity of uptake of asialoorosomucoid in the liver (86%) , as opposed to other tissues. B. Biotinylation of asialoorosomucoid clearing agent and orosomucoid control. 100 μl of 0.2 M sodium carbonate buffer, pH 9.2, was added to 2 mg (in 1.00 ml PBS) of PIP-125-labeled orosomucoid and to 2 mg PIP-125-labeled asialoorosomucoid. 60 μl of a 1.85 mg/ml solution of NHS-amino caproate biotin in DMSO was then added to each compound. The reaction mixtures were vortexed and allowed to sit at room temperature for 45 minutes. The material was purified by size exclusion column chromatography (PD-10, Pharmacia) and eluted with PBS. 1.2 ml fractions were taken, with fractions 4 and 5 containing the majority of the applied radioactivity (>95%) . Streptavidin- agarose beads (Sigma S-1638) or -pellets were washed with PBS, and 20 μg of each biotinylated, radiolabeled protein was added to 400 μl of beads and 400 μl of PBS, vortexed for 20 seconds and centrifuged at 14,000 rpm for 5 minutes. The supernatant was removed and the pellets were washed with 400 μl PBS. This wash procedure was repeated twice more, and the combined supernatants were assayed by placing them in a dosimeter versus their respective pellets. The values are shown below in Table 4. C. Protein-Streptavidin Binding in vivo. Biotin- asialoorosomucoid was evaluated for the ability to couple with circulating LU-10-StrAv conjugate in vivo and to remove it from the blood. Female BALB/c mice (20-25 g) were injected i.v. with 200 μg LU-10-StrAv conjugate. Clearing agent (200 μl PBS - group 1; 400 μg non-biotinylated asialoorosomucoid - group 2; 400 μg biotinylated asialoorosomucoid - group 3; and 200 μg biotinylated asialoorosomucoid- group 4) was administered at 25 hours following conjugate administration. A fifth group received PIP-I-131-LU- 10-StrAv conjugate which had been saturated prior to injection with biotin - group 5. The 400 μg dose constituted a 10:1 molar excess of clearing agent over the initial dose of LU-10-StrAv conjugate, while the 200 μg dose constituted a 5:1 molar excess. The saturated PIP-I-131-LU-10-StrAv conjugate was produced by addition of a 10-fold molar excess of D-biotin to 2 mg of LU-10-StrAv followed by size exclusion purification on a G-25 PD-10 column. Three mice from each group were serially bled, as described above, at 0.17, 1, 4 and 25 hours (pre- injection of clearing agent), as well as at 27, 28, 47, 70 and 90 hours. Two additional animals from each group were sacrificed at 2 hours post-clearing agent administration and limited biodistributions were performed. The blood clearance data are shown in Figure 7. These data indicate that circulating LU-10-StrAv radioactivity in groups 3 and 4 was rapidly and significantly reduced, in comparison to those values obtained in the control groups 1, 2 and 5. Absolute reduction in circulating antibody-streptavidin conjugate was approximately 75% when compared to controls. Biodistribution data are shown in tabular form in Figure 8. The biodistribution data show reduced levels of conjugate for groups 3 and 4 in all tissues except the liver, kidney and intestine, which is consistent with the processing and excretion of radiolabel associated with the conjugate after complexation with biotinylated asialoorosomucoid. Furthermore, residual circulating conjugate was obtained from serum samples by cardiac puncture (with the assays conducted in serum + PBS) and analyzed for the ability to bind biotin (immobilized biotin on agarose beads) , an indicator of functional streptavidin remaining in the serum. Group 1 animal serum showed conjugate radiolabel bound about 80% to immobilized biotin. Correcting the residual circulating radiolabel values by multiplying the remaining percent injected dose (at 2 hours after clearing agent administration) by the remaining percent able to bind immobilize biotin (the amount of remaining functional conjugate) leads to the graph shown in Figure 9. Administration of 200 μg biotinylated asialoorosomucoid resulted in a 50-fold reduction in serum biotin-binding capacity and, in preliminary studies in tumored animals, has not exhibited cross-linking and removal of prelocalized LU-10-StrAv conjugate from the tumor. Removal of circulating targeting moiety-anti-ligand without diminishing biotin-binding capacity at target cell sites, coupled with an increased radiation dose to the tumor resulting from an increase in the amount of targeting moiety-anti-ligand administered, results in both increased absolute rad dose to tumor and diminished toxicity to non-tumor cells, compared to what is currently achievable using conventional radioimmunotherapy. A subsequent experiment was executed to evaluate lower doses of asialoorosomucoid-biotin. In the same animal model, doses of 50, 20 and 10 μg asialoorosomucoid-biotin were injected at 24 hours following administration of the LU-10-StrAv conjugate. Data from animals serially bled are shown in Figure 10, and data from animals sacrificed two hours after clearing agent administration are shown in Figures 11A (blood clearance) and 11B (serum biotin-binding) , respectively. Doses of 50 and 20 μg asialoorosomucoid-biotin effectively reduced circulating LU-10-StrAv conjugate levels by about 65% (Figure 11A) and, after correction for binding to immobilized biotin, left only 3% of the injected dose in circulation that possessed biotin-binding capacity, compared with about 25% of the injected dose in control animals (Figure 11B) . Even at low doses (approaching 1:1 stoichiometry with circulating LU-10- StrAv conjugate) , asialoorosomucoid-biotin was highly effective at reducing blood levels of circulating streptavidin-containing conjugate by an m vivo complexation that was dependent upon biotin-avidin interaction. Example XIV Tumor uptake of PIP-Biocytin PIP-Biocytin, as prepared and described in Example VII above, was tested to determine the fate thereof .in vivo. The following data are based on experimentation with tumored nude mice (100 mg LS-180 tumor xenografts implanted subcutaneously 7 days prior to study) that received, at time 0, 200 μg of 1-125 labeled NR-LU-10- Streptavidin conjugate (950 pmol) , as discussed in Example XI above. At 24 hours, the mice received an i.v. injection of PIP-I-131-biocytin (40 μCi) and an amount of cold carrier PIP-I-127 biocytin corresponding to doses of 42 μg (69,767 pmol) , 21 μg (34,884 pmol), 5.7 μg (9468 pmol) , 2.85 μg (4734 pmol) or 0.5 μg (830 pmol). Tumors were excised and counted for radioactivity 4 hours after PIP-biocytin injection. The three highest doses produced PIP-biocytin tumor localizations of about 600 pmol/g. Histology conducted on tissues receiving the two highest doses indicated that saturation of tumor-bound streptavidin was achieved. Equivalent tumor localization observed at the 5.7 μg dose is indicative of streptavidin saturation as well. In contrast, the two lowest doses produced lower absolute tumor localization of PIP- biocytin, despite equivalent localization of NR-LU-10- Streptavidin conjugate (tumors in all groups averaged about 40% ID/G for the conjugate) . The lowest dose group (0.5 μg) exhibited high efficiency tumor delivery of PIP-I-131-biocytin, which efficiency increased over time. A peak uptake of 85.0 % ID/G was observed at the 120 hour time point (96 hours after administration of PIP-biocytin) . Also, the absolute amount of PIP-biocytin, in terms of % ID, showed a continual increase in the tumor over all of the sampled time points. The decrease in uptake on a % ID/G basis at the 168 hour time point resulted from significant growth of the tumors between the 120 and 168 hour time points. In addition, the co-localization of NR-LU-10- Streptavidin conjugate (LU-10-StrAv) and the subsequently administered PIP-Biocytin at the same tumors over time was examined. The localization of radioactivity at tumors by PIP-biocytin exhibited a pattern of uptake and retention that differed from that of the antibody-streptavidin conjugate (LU-10- StrAv) . LU-10-StrAv exhibited a characteristic tumor uptake pattern that is equivalent to historical studies of native NR-LU-10 antibody, reaching a peak value of 40% ID/G between 24 and 48 hours after administration. In contrast, the PIP-Biocytin exhibited an initial rapid accretion in the tumor, reaching levels greater than those of LU-10-StrAv by 24 hours after PIP-Biocytin administration. Moreover, the localization of PIP-Biocytin continued to increase out to 96 hours, when the concentration of radioactivity associated with the conjugate has begun to decrease. The slightly greater amounts of circulating PIP-Biocytin compared to LU-10-StrAv at these time points appeared insufficient to account for this phenomenon. The ratio of PIP-Biocytin to LU-10-StrAv in the tumor increased continually during the experiment, while the ratio in the blood decreased continually. This observation is consistent with a process involving continual binding of targeting moiety- containing conjugate (with PIP-Biocytin bound to it) from the blood to the tumor, with subsequent differential processing of the PIP-Biocytin and the conjugate. Since radiolabel associated with the streptavidin conjugate component (compared to radiolabel associated with the targeting moiety) has shown increased retention in organs of metabolic processing, PIP-Biocytin associated with the streptavidin appears to be selectively retained by the tumor cells. Because radiolabel is retained at target cell sites, a greater accumulation of radioactivity at those sites results. The for PIP-Biocytin is over twice that of the conjugate (4.27 compared to 1.95, where AUC means "area under the curve") . Further, the absolute A ~~ C tLm „ for PIP-Biocytin is nearly twice that of the conjugate (9220 compared to 4629) . Consequently, an increase in radiation dose to tumor was achieved. Example XV Synthesis of DOTA-Biotin Conjugates A. Synthesis of Nitro-Benzyl-DOTA. 1. Scheme 1 The synthesis of aminobenzyl-DOTA was conducted substantially in accordance with the procedure of McMurry et al., Biocon ugate Chem. , 3.: 108-117, 1992. The critical step in the prior art synthesis is the intermolecular cyclization between disuccinimidyl N- (tert-butoxycarbonyl)iminodiacetate and N-(2- a inoethyl)-4-nitrophenyl alaninamide to prepare 1- (tert-butoxycarbonyl)-5-(4-nitrobenzyl)-3,6,11-trioxo- 1,4,7,10-tetraazacyclododecane. In other words, the critical step is the intermolecular cyclization between the bis-NHS ester and the diamine to give the cyclized dodecane. McMurry et al. conducted the cyclization step on a 140 mmol scale, dissolving each of the reagents in 100 ml DMF and adding via a syringe pump over 48 hours to a reaction pot containing 4 liters dioxane. A 5x scale-up of the McMurry et al. procedure was not practical in terms of reaction volume, addition rate and reaction time. Process chemistry studies revealed that the reaction addition rate could be substantially increased and that the solvent volume could be greatly reduced, while still obtaining a similar yield of the desired cyclization product. Consequently on a 30 mmol scale, each of the reagents was dissolved in 500 ml DMF and added via addition funnel over 27 hours to a reaction pot containing 3 liters dioxane. The addition rate of the method employed involved a 5.18 mmol/hour addition rate and a 0.047 M reaction concentration. 2. Scheme 2 - Triamide Route (Figure 12) Compound (I. To a stirred ethylenediamine (190 ml; 2.84 mole) was added 21 g (70.87 m mole) of N-BOC-p- nitrophenylglycinate through a powder additional funnel in small portions. The mixture was stirred at room temperature under nitrogen for 5 hours. Excess of ethylenediamine was removed under vacuum and the resulting residue was dissolved in 100 ml water. The aqueous solution was adjusted to pH 10.5 with 2N NaOH and then extracted continuously with CH 2 C1 2 overnight. The CH 2 C1 2 layer was separated and stored at 4°C overnight. Crystalline material formed and it was removed by filtration. (It was salt complex of ethylenediamine and p-nitrophenol) . The filtrate was washed with saturated sodium carbonate (3 x 60 ml) to remove the remaining ethylene diammonium salt. The CH 2 C1 2 layer was dried over sodium sulfate, filtered and evaporated to give 13.423 g compound (I). (87.2 %) l HNMR (CD 3 CN, δ) : 6.70 and 5.65 (2bs, NH) , 3.58 (d, 2H, CH 2 CO, 3.13 (m, 2H, NCH 2 ) , 2.64 (t, 2H, NCH 2 ) , 1.40 (s, 9H, t-butyl) 13 CNMR (CD 3 CN, ppm) : 176.0, 171.2, 80.2, 44.9, 43.1, 42.4, 28.6. Compound (II. To a solution of 13.32g (61.26 mmole) of compound (I) and 2.13 ml (12.17 mmole) of diisopropylethylamine in 84 ml dry MeOH was added dropwise 8.73 ml (10.454 g; 73.47 mmole) of ethyltrifluoroacetate at room temperature under nitrogen. The reaction mixture was stirred at room temperature overnight. Solvent was removed under vacuum to give a crude product. This crude product was purified by flash column silica gel chromatography (eluted with EtOAc) to give 15.378 g compound (II). (92.5%) 'HNMR (CDC1 3 , δ) : 7.93, 6.87 and 5.24 (3bS, 3H, NH) , 3.79 (d, 2H, CH 2 C0) , 3.50 (m, 4H, NaCH 2 ) , 1.46 (s, 9H, t-butyl) . 13 CNMR (CDC1 3 , ppm): 171.76, 159.34, 158.60, 157.86, 157.12, 156.50, 124.50, 118.77, 113.04, 107.31, 80.34, 43.94, 40.61, 38.28 and 27.84. Compound (III) To a suspension of 4.204g (20 mmole) of p- nitrophenylalanine in 200 ml t-butylacetate was added 3.154g (1.1 eq) of 70% HC10 4 . The mixture was stirred at room temperature for 7 days. The reaction mixture was extracted with ice-cold 0.5N HCl (4 x 75 ml) . This aqueous extraction was neutralized immediately with solid sodium bicarbonate at 0°C. The resulting precipitate was filtered off and the filtrate was extracted with EtOAC (5 x 75 ml) . The EtOAC extraction was dried over sodium sulf te, filters and evaporated to give 3.674 g compound (III) in a form of a pale yellow solid. (70%) J HNMR (CDC1 3 , 5): 8.188 and 7.42 (2d, 4H, ArH) , 3.70 (t, 1H, CH) , 3.08 (m, 2H, CH 2 ) , 1.43 (s, 9H, t-butyl) . Compound (IV) To a solution of 1.093g (4.11 mmole) of compound (III) and 752 μl (1.05 eq) of diisopropylethylamine in 25 ml dry CH 2 C1 2 was added slowly a solution of 1.643 g (4.52 mmole) of iodoacetic anhydride in 8 ml dry CH 2 C1 2 at 0°C under nitrogen. The mixture was stirred at room temperature for 3 hours. Solvent was removed under reduced pressure and the resulting crude product was purified by flash column silica gel chromatography (eluted with CH 2 Cl 2 :EtOAc = 96:4) to give 16.3 g compound (IV). (90%) J HNMR (CDC1 3 , δ) : 8.17 and 7.37 (2d, 4H, ArH), 6.53 (d, 1H, NH) , 4.76 (q, 1H, CH) , 3.69 (d, 2H, CH 2 I) , 3.24 (m, 2H, CH 2 ) , 1.44 (s, 9H, t- butyl) . 13 CNMR (CDC1 3 , ppm): 170.03, 167.05, 147.64, 144.17, 130.91, 123.97, 83.85, 54.09, 37.77, 28.11 and -1.47. Compound (V) To a suspension of 65 mg (1.63 mmole) of 60% oil dispersion of NaH in 6 ml dry THF (freshly distilled) at -5°C under argon was added a solution of 440.8 mg (1.63 mmole) of compound (II) in 1.6 ml dry THF. The mixture was stirred at room temperature for 30 minutes and then cooled back to -5°C. To this mixture was added dropwise a solution of 2 m mole of compound (IV) in 2 ml dry THF. The mixture was stirred at room temperature overnight and then diluted with EtOAc. The EtOAc layer was washed with 0.5N HCl (ice-cold) , H 2 0, 5% NaHC0 3 , H 2 0, and brine. The separated EtOAc layer was dried over sodium sulfate, filtered and evaporated to give a crude product. This crude product was purified by flash column silicon gel chromatography (eluted with CH 2 C1 2 :t-BuOH:Et 3 N = 92:8:0.5) to give 423 mg compound (V) . (42%) . 'HNMR (CDC1 3 , δ) : 8.18 and 7.42 (2m, 4H, ArH), 6.98-6.72 (m, 2H, NH) , 5.19 (bs, 1H, NH) , 4.71 ( , 1H, CH) , 4.14- 3.48 (m, 8H, CH 2 ) , 3.20 (m, 2H, CH 2 Ar) , 1.45 (s, 18H, t-butyl) . Mass spectrum (FAB) : m/e 620 (MH + ) , 564 (MH 2 + -t-butyl) . Compound (VI) To a solution of 5.035 g (8.13 mmole) of compound (V) in 48 ml dry CH 2 C1 2 at 0°C was added 72 ml trifluoroacetic acid. The reaction mixture was stirred at room temperature and the progress of the reaction was monitored by TLC (C-18, MeOH = H 2 0 = 7/3). After 3 hours, another 10 ml of trifluoroacetic acid was added to the reaction mixture and the reaction was allowed to continue for one more hour. Solvent was removed under reduced pressure and the resulting residue was co-evaporated twice with dry CH 3 CN, once with dry toluene to give 5.211 g compound (VI) with 1.56 molar ratio of TFA. (100%) J HNMR (CD 3 CN, δ) 8.13 and 7.46 (2m, ArH), 7.23-6.80 (m, NH) , 4.72 (m, CH) , 4.04 and 3.60-3.28 (m & , CH 2 ) , 3.12 ( , CH 2 Ar) . Compound (VII) To a suspension of 638 mg (1 eq) NaHC0 3 in 850 ml dry DMF at 50°C under argon was injected simultaneously via a syringe pump a solution of 4.871 g (7.6 mmole) of compound (VI) in 50 ml dry DMF, a solution of 3.867 g (1.15 eq) BOP in 50 ml dry DMF, a solution of 3.867 g (1.15 eq) BOP in 50 ml dry DMF and a solution of 3.3 ml (19 mmole) of diisopropylethylamine in 50 ml dry DMF over a period of 1 hour. The reaction mixture was continued to stir at the same temp for 1 hour at the end of the addition and then room temperature overnight. Aqueous HOAc was added to the mixture and the solvent was removed under high vacuum to give a crude residue. Methanol (30 ml) was added to this residue and the resulting precipitate was filtered, washed with MeOH and dried under vacuum to give 2.079 g compound (VII). (61.4%). - 'HNMR (DMS0-d 6 , <5) : 9.26 (d, NH) , 8.44 (m, NH) , 8.15 and 7.50 (2m, ArH), 6.80 (m, NH) , 4.50 (q, CH) , 4.38 and 4.29 (2s, CH 2 ) , 4.02- 2.60 (m, CH 2 ) . Compound (VIII) A suspension of 2.129 g (4.67 mmole of compound (VII), in 400 ml NH 4 0H (cone.) and 400 ml MeOH was stirred at room temperature in a tightly sealed flask overnight. The mixture was filtered, and the filter was washed with MeOH. The combined filtrate and wash was evaporated to give 2.15 g compound (VIII). (99%) ! HNMR (DMSO-d 6 + D 2 0, δ) : 8.12 and 7.50 (2M, 4H, ArH), 4.48 (m, 1H, CH 2 ) , 3.92-2.60 ( , CH 2 ) . Mass spectrum (FAB) : m/e 350 (MH + ) . Compound (IX) To a suspension of 1.675 g (3.62 mmole) of compound (VIII) in 18 ml dry THF was added slowly 39.8 ml of 1M BH 3 in THF at 0°C under argon. The mixture became clear and it was heated at reflux temperature for 24 hours. The mixture was cooled to 0°C and to it was added slowly 3 ml MeOH to quench the excess BH 3 . The mixture was then stirred at room temperature for 10 minutes. Solvent was removed under reduced pressure and the resulting residue was suspended in 100 ml dry MeOH. To this suspension at 0°C was saturated with HC1 gas and then the clear mixture was heated at reflux temperature for 1 day. Solvent was removed under reduced pressure to give 1.62 g crude product. This crude product was recrystallized in MeOH to give 490.2 mg compound (IX) as the first crop. The filtrate of the recrystallization solution was concentrated and purified by flash column C-18 reverse phase chromatography (eluted with H 2 0: MeOH:TFA = 80:20:0.1) to give further 501.6 mg compound (IX) . (65.7%) *HNMR (D 2 0, δ) : 8.25 and 7.53 (2d, 4H, ArH), 3.41 ( , 1H, CH) , 3.26-2.89 (m, 16H, CH 2 ) . Mass spectrum (FAB) : m/e 308 (MH + ) . 3. Scheme 3 (Direct Peptide Cyclization - Figures 13 and 14; Reverse Direct Peptide Cyclization - Figure 15) The schemes of Figures 13 and 14 are particularly advantageous because they require relatively few synthetic steps, and with the possible exception of the final step, no chromatographic purifications are required. Description of scheme of Figure 14 N-BOC-4-nitro-L-phenylalanine (1) To a stirred suspension of 50.45 g (240 mmole) of 4-nitro-L-phenylalanine in 140 ml water was added a solution of 65.11 g (264.4 mmole; 1.1 eq) of BOC-ON in 140 ml dioxane and 53 ml (380.3 mmole; 1.58 eq) of Et 3 N. The gradually cleared solution was stirred at room temperature for 4 hours. The reaction mixture was poured into 410 ml water and extracted with ether (5 x 330 ml) . The aqueous layer was acidified to pH 1.15 with 6N HC1 and then extracted with EtOAc (5 x 330 ml) . The EtOAc extracts were pooled and dried over with MgS0 4 , filtered and evaporated under reduced pressure to give a crude residue. This residue was dissolved in 300 ml ether, the solvent was removed under reduced pressure to give 67.61 g (90.8%) product (1) as a pale yellow foam. 1 HNMR (CDC1 3 , δ) = 8.17 & 7.38 (d & d, 4H) , 5.15 & 4.55 (m & m, 2H) , 3.20 (m, 2H) , 1.38 (2s, 9H) . N-BOC-4-nitrophenylalanine N-hvdroxysuccinimide ester 111 To a stirred solution of 67.61 g (217.9 mmole) of (1) and 48.79 g (236.5 mmole) of dicyclohexylcarbodiimide in 1083 ml dry dioxane was added 25.82 g (224.3 mmole) of N-hydroxysuccinimide. The reaction mixture was stirred at room temperature under nitrogen for 17 hours and then filtered to remove the DCU. The filtrate was evaporated under reduced pressure and the resulting residue was suspended in 865 ml 2-propanol heated at 50°C for 10 minutes. When cooled to room temperature, the precipitate was filtered, washed twice with ice-cold 2-propanol and dried under vacuum to give 78.21 g (2) (88.1%) as a pale yellow solid. ^NMR (CDC1 3 , δ) = 8.20 & 7.50 (d & d, 4H) , 4.95 (m, 2H, 3.37 (m, 2H) , 2.88 (s, 4H) , 1.43 (s, 9H) . N-BOC-p-nitrophenylalanylglycylglvcylglycine (3) To a stirred solution of 78.0 g (191.5 mmole) of (3) in 342 ml DMF was added a solution of 48.37 g (255.5 mmole) of triglycine and 28.53 g (339.6 mmole) of NaHC0 3 in 677 ml of water. The reaction mixture was stirred at room temperature for 18 hours then poured into 1460 ml water. This solution was adjusted to pH 1.15 with 6N HC1 and then stored at 4°C for 9h. The precipitate formed was filtered off, washed with ice- cold DMF/H 2 0 (8/92, V/V; pH 1.15) (100 ml) and ether (2 x 300 ml) . The precipitate was dried at 65°C under high vacuum to give 79.14 g (85.8%) of (3) as a pale yellow solid 1 HNMR (DMSO-d6, δ) = 8.18 (m, 5H) , 7.57 (d, 2H) , 7.10 (d, 1H) , 4.28 (m, 1H) , 3.76 (m, 6H) , 3.02 (m, 2H) , 1.28 (s, 9H) . p-nitrophenylalanylglycylglycylglvcine (4) To a stirred suspension of 79 g (164 mmole) of (4) in 400 ml CH 2 C1 2 at 0°C was added 400 ml trifluoroacetic acid. The reaction mixture was stirred at room temperature for 30 minutes. Solvent was removed under reduced pressure and the resulting residue was co-evaporated twice with CH 2 C1 2 . The foamy product was further dried under high vacuum for 3 days to give 104.4 g (quantitative) of (4) as a 2.24 TFA salt. ! HNMR (CD 3 CN, <S) = 8.18 & 7.50 (d & d, ArH), 7.57, 7.33 & 7.09 (t, t, & t, NH) , 4.28 (t, CH) , 3.85 (m, CH 2 ) , 3.28 (m, CH 2 ) . 2-(p-nitrobenzyl)-3.6,9-12-tetraoxo-l.4,7,10- tetraazacyclododecane (5) To a mechanically stirred solution of 2800 ml dry DMF and 13.6 ml diisopropylethylamine at 85°C under nitrogen was co-injected a solution of 50.91 g (80 mmole) of (4) in 480 ml dry DMF, a solution of 46.02 g (1.3 eq.) of benzotriazolyloxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP) in 480 ml dry DMF and a solution of 44.8 ml (3.2 eq.) diisopropylethylamine in 440 ml dry DMF through HPLC pumps at a flow rate of 0.6 ml/min. After the completion of the co-injection (14.5 hours), the reaction mixture was allowed to continue to stir at 85°C for 1 hour. The mixture was cooled to 4°C and the precipitate was filtered, washed with ether 3 times and dried under vacuum to give 16.47 g (56.7%) crude (5) . Due to the very poor solubility to most of solvent (except TFA) , (5) was used directly in the next reaction without further purification. 2-(p-nitrobenzyl) -1.4.7.10-tetraazacvclododecane (6) To a stirred suspension of 16.4 g (45.13 mmole) of (6) in 540 ml dry THF was added slowly 615.5 ml of 1 M BH 3 in THF at 0°C. The reaction mixture was heated at reflux for 1 day under nitrogen. The reaction was quenched by slow addition of 110 ml dry MeOH to the mixture at 0°C solvent was removed under reduced pressure and the resulting residue was suspended in 1000 ml dry MeOH. To this suspension at 0°C was saturated with HCl gas and then heated at reflux for 1 day. The still hot reaction solution was decanted off and the solvent was removed under reduced pressure to give 32.3 g crude (6). This crude (6) was recrystallized in MeOH to give 3.574 g off-white (6) as a solid. The filtrate was concentrated and purified by Bio-Rex 70 weak cation exchange resin (500 ml bed volume) eluted with 0.02N, NH 4 0Ac (pH 7) with gradual increase of solvent B (NH 4 OH/MeOH/H 2 0 = 3/3/4) . The fractions which contained the product were pooled and concentrated to dryness. The dried product was dissolved in 400 ml MeOH and then saturated with HCl. After the solvent removal, 4.773 g (6) was obtained as a trihydrochloride salt. Yield: 71%, X H-NMR (D 2 0, δ) : 8.17 and 7.46 (d & d, 4H, ArH), 3.35 (m, 1H, CH) , 2.90 (m, 16H, CH 2 ) . The schemes of Figures 14 and 15 are alternative methods for producing nitro-benzyl-DOTA. B. Synthesis of a D-alanine-linked conjugate with a preserved biotin carboxy moiety. A reaction scheme to form a compound of the following formula is discussed below. first coupling D-alanine (Sigma Chemical Co.) to biotin-NHS ester. The resultant biotinyl-D-alanine was then activated with l-(3-dimethylaminopropyl)-3- ethyl-carbodii ide hydrochloride (EDCI) and N- hydroxysuccinimide (NHS) . This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110 * C for 30 minutes. The solution was cooled to 23 * C and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D- alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield. NHS (10 mg, 0.08 mmol) and EDCI (15 mg, 0.07 mmol) were added to a solution of biotinyl-D-alanine (27 mg, 0.08 mmol) in DMF (1 ml) . The solution was stirred at 23'C for 60 hours, at which time TLC analysis indicated conversion of the carboxyl group to the N- hydroxy succinimidyl ester. Pyridine (0.8 ml) was added followed by DOTA-aniline (20 mg, 0.04 mmol). The mixture was heated momentarily at approximately 100"C, then cooled to 23 * C and evaporated. The product, DOTA-aniline-D-alanyl-biotinamide was purified by preparative HPLC. C. Synthesis of N-hvdroxyethyl-linked conjugate. Iminodiacetic acid dimethyl ester is condensed with biotin-NHS-ester to give biotinyl dimethyl iminodiacetate. Hydrolysis with one equivalent of sodium hydroxide provides the monomethyl ester after purification from under and over hydrolysis products. Reduction of the carboxyl group with borane provides the hydroxyethyl amide. The hydroxyl group is protected with t-butyl-dimethyl-silylchloride. The methyl ester is hydrolysed, activated with EDCI and condensed with DOTA-aniline to form the final product conjugate. D . Synthesis of N-Me-LC-DOTA-biotin . A reaction scheme is shown below. Esterification of 6-aminocaproic acid (Sigma Chemical Co.) was carried out with methanolic HCl. Trifluoroacetylation of the amino group using trifluoroacetic anhydride gave N-6-(methylcaproyl)- trifluoroacetamide. The amide nitrogen was methylated using sodium hydride and iodomethane in tetrahydrofuran. The trifluoroacetyl protecting group was cleaved in acidic methanol to give methyl 6- methyla ino-caproate hydrochloride. The amine was condensed with biotin-NHS ester to give methyl N- methyl-caproylamido-biotin. Saponification afforded the corresponding acid which was activated with EDCI and NHS and, in situ, condensed with DOTA-aniline to give DOTA-benzylamido-N-methyl-caproylamido-biotin. 1. Preparation of methyl 6-aminocaproate hydrochloride. Hydrogen chloride (gas) was added to a solution of 20.0 g (152 mmol) of 6-aminocaproic acid in 250 ml of methanol via rapid bubbling for 2-3 minutes. The mixture was stirred at 15-25"C for 3 hours and then concentrated to afford 27.5 g of the product as a white solid (99%) : H-NMR (DMSO) 9.35 (1 H, broad t) , 3.57 (3H, s) , 3.14 (2H, quartet), 2.28 (2H, t) , 1.48 (4H, multiplet) , and 1.23 ppm (2H, multiplet) . 2. Preparation of N-6-(methylcaproyl)- trifluoroacetamide. To a solution of 20.0 g (110 mmol) of methyl 6-aminocaproate hydrochloride in 250 ml of dichloromethane was added 31.0 ml (22.2 mmol) of triethylamine. The mixture was cooled in an ice bath and trifluoroacetic anhydride (18.0 ml, 127 mmol) was added over a period of 15-20 minutes. The mixture was stirred at 0-10'C for 1 hour and concentrated. The residue was diluted with 300 ml of ethyl acetate and saturated aqueous sodium bicarbonate (3 x 100 ml) . The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to afford 26.5 g of the product as a pale yellow oil (100%) : H-NMR (DMSO) 3.57 (3H, Ξ) , 3.37 (2H, t) , 3.08 (1.9H, quartet, N-CH 3 ) , 2.93 (1.1H, s, N-CH 3 ) , 2.30 (2H, t) , 1.52 (4H, multiplet), and 1.23 ppm (2H, multiplet) . 3. Preparation of methyl 6-N-methylamino-caproate hydrochloride. To a solution of 7.01 g (29.2 mmol) of N-6-(methylcaproyl) -trifluoroacetamide in 125 ml of anhydrous tetrahydrofuran was slowly added 1.75 g of 60% sodium hydride (43.8 mmol) in mineral oil. The mixture was stirred at 15-25 * C for 30 minutes and then 6.2 g (43.7 mmol) of iodomethane was added. The mixture was stirred at 15-25°C for 17 hours and then filtered through celite. The solids were rinsed with 50 ml of tetrahydrofuran. The filtrates were combined and concentrated. The residue was diluted with 150 ml of ethyl acetate and washed first with 5% aqueous sodium sulfite (2 x 100 ml) and then with 100 ml of 1 N aqueous hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to afford a yellow oily residue. The residue was diluted with 250 ml of methanol and then hydrogen chloride (gas) was rapidly bubbled into the mixture for 2-3 minutes. The resultant mixture was refluxed for 18 hours, cooled and concentrated. The residue was diluted with 150 ml of methanol and washed with hexane (3 x 150 ml) to remove mineral oil previously introduced with NaH. The methanol phase was concentrated to afford 4.91 g of the product as a yellow oil (86%) : H-NMR (DMSO) 8.80 (2H, broad s) , 3.58 (3H, s) , 2.81 (2H, multiplet), 2.48 (3H, s) , 2.30 (2H, t) , 1.52 (4H, multiplet), and 1.29 ppm (2H, multiplet) . 4. Preparation of methyl 6-(N-methylcaproylamido- biotin. N-hydroxysuccinimidyl biotin (398 mg, 1.16 mmol) was added to a solution of methyl 6-(N-methyl) aminocaproate hydrochloride (250 mg, 1.28 mmol) in DMF (4.0 ml) and triethylamine (0.18 ml, 1.28 mmol). The mixture was heated in an oil bath at 100'C for 10 minutes. The solution was evaporated, acidified with glacial acetic acid and evaporated again. The residue was chromatographed on a 25 mm flash chromatography column manufactured by Ace Glass packed with 50 g silica (EM Science, Gibbstown, New Jersey, particle size 0.40-0.63 mm) eluting with 15% MeOH/EtOAc. The product was obtained as a yellow oil (390 mg) in 79% yield. 5. Preparation of 6-(N-methyl-N-biotinyl) amino caproic acid. To a solution of methyl 6-(N-methyl- caproylamido-biotin (391 mg, 1.10 mmol) in methanol (2.5 ml) was added a 0.95 N NaOH solution (1.5 ml). This solution was stirred at 23 β C for 3 hours. The solution was neutralized by the addition of 1.0 M HCl (1.6 ml) and evaporated. The residue was dissolved in water, further acidified with 1.0 M HCl (0.4 ml) and evaporated. The gummy solid residue was suspended in water and agitated with a spatula until it changed into a white powder. The powder was collected by filtration with a yield of 340 mg. 6. Preparation of DOTA-benzylamido-N-methyl- caproylamido-biotin. A suspension of 6-(N-methyl-N- biotinyl)amino caproic acid (29 mg, 0.08 mmol) and N- hydroxysuccmimide (10 mg, 0.09 mmol) in DMF (0.8 ml) was heated over a heat gun for the short time necessary for the solids to dissolve. To this heated solution was added EDCI (15 mg, 0.08 mmol). The resultant solution was stirred at 23 * C for 20 hours. To this stirred solution were added aminobenzyl-DOTA (20 mg, 0.04 mmol) and pyridine (0.8 ml). The mixture was heated over a heat gun for 1 minute. The product was isolated by preparative HPLC, yielding 3 mg. E. Synthesis of a bis-DOTA conjugate with a preserved biotin carboxy group. A reaction scheme is shown below. MeOH H0-C0-(CH,) 5 -Br > CH,-0-CO-(CH 2 ) 5 -Br HCl(g) C- j -CO-NH-(CH j )J-CO-O-CHJ 1. Preparation of methyl 6-bromocaproate (methyl 6-bromohexanoate) . Hydrogen chloride (gas) was added to a solution of 5.01 g (25.7 mmol) of 6-bromocaproic acid in 250 ml of methanol via vigorous bubbling for 2-3 minutes. The mixture was stirred at 15-25 * C for 3 hours and then concentrated to afford 4.84 g of the product as a yellow oil (90%) : H-NMR (DMSO) 3.58 (3H, s) , 3.51 (2H, t) , 2.29 (2H, t) , 1.78 (2H, pentet) , and 1.62-1.27 ppm (4H, m) . 2. Preparation of N,N-bis-(methyl 6-hexanoyl)- amine hydrochloride. To a solution of 4.01 g (16.7 mmol) of N-(methyl 6-hexanoyl)-trifluoroacetamide (prepared in accordance with section D.2. herein) in 125 ml of anhydrous tetrahydrofuran was added 1.0 g (25 mmol) of 60% sodium hydride in mineral oil. The mixture was stirred at 15-25'C for 1 hour and then 3.50 g (16.7 mmol) of methyl 6-bromocaproate was added and the mixture heated to reflux. The mixture was stirred at reflux for 22 hours. NMR assay of an aliquot indicated the reaction to be incomplete. Consequently, an additional 1.00 g (4.8 mmol) of methyl 6-bromocaproate was added and the mixture stirred at reflux for 26 hours. NMR assay of an aliquot indicated the reaction to be incomplete. An additional 1.0 g of methyl 6-bromocaproate was added and the mixture stirred at reflux for 24 hours. NMR assay of an aliquot indicated the reaction to be near complete. The mixture was cooled and then directly filtered through celite. The solids were rinsed with 100 ml of tetrahydrofuran. The filtrates were combined and concentrated. The residue was diluted with 100 ml of methanol and washed with hexane (3x 100 ml) to remove the mineral oil introduced with the sodium hydride. The methanol phase was treated with 6 ml of 10 N aqueous sodium hydroxide and stirred at 15- 25 * C for 3 hours. The mixture was concentrated. The residue was diluted with 100 ml of deionized water and acidified to pH 2 with concentrated HCl. The mixture was washed with ether (3 x 100 ml) . The aqueous phase was concentrated, diluted with 200 ml of dry methanol and then hydrogen chloride gas was bubbled through the mixture for 2-3 minutes. The mixture was stirred at 15-25"C for 3 hours and then concentrated. The residue was diluted with 50 ml of dry methanol and filtered to remove inorganic salts. The filtrate was concentrated to afford 1.98 g of the product as a white solid (38%) : H-NMR (DMSO) 8.62 (2H, m) 3.58 (6H, s) , 2.82 (4H, m) 2.30 (4H, t) , 1.67-1.45 (8H, m) and 1.38-1.22 ppm (4H, m) . 3. Preparation of N,N'-bis-(methyl 6-hexanoyl)- biotinamide. To a solution of 500 mg (1.46 mmol) of N-hydroxysuccinimidyl biotin in 15 ml of dry dimethyl- formamide was added 600 mg (1.94 mmol) of N,N-bis- (methyl 6-hexanoyl) amine hydrochloride followed by 1.0 ml of triethylamine. The mixture was stirred at 80- 85 * C for 3 hours and then cooled and concentrated. The residue was chromatographed on silica gel, eluting with 20% methanol/ethyl acetate, to afford 620 mg of the product as a near colorless oil (85%) : H-NMR (CDC1 3 ) 5.71 (1H, s) , 5.22 (1H, s) , 4.52 (1H, m) , 4.33 (1H, m) , 3.60 (3H, s) , 3.58 (3H, s) , 3.34-3.13 (5H, m) , 2.92 (1H, dd) , 2.75 (1H, d) , 2.33 (6H, m) and 1.82-1.22 ppm (18H, m) ; TLC-R f 0.39 (20:80 methanol/ ethyl acetate). 4. Preparation of N,N-bis-(6-hexanoyl) - biotinamide. To a solution of 610 mg (0.819 mmol) of N,N-bis-(methyl 6-hexanoyl) -biotinamide in 35 ml of methanol was added 5.0 ml of IN aqueous sodium hydroxide. The mixture was stirred at 15-25 * C for 4.5 hours and then concentrated. The residue was diluted with 50 ml of deionized water acidified to pH 2 with IN aqueous hydrochloric acid at 4 * C. The product, which precipitated out as a white solid, was isolated by vacuum filtration and dried under vacuum to afford 482 mg (84%) : H-NMR (DMSO) 6.42 (1H, s) , 6.33 (1H, s) , 4.29 (1H, m) , 4.12 (1H, m) , 3.29-3.04 (5H, m) , 2.82 (1H, dd) , 2.57 (1H, d) , 2.21 (6H, m) and 1.70- 1.10 ppm (18H, m) . 5. Preparation of N' ,N'-bis-(N-hydroxy- succinimidyl 6-hexanoyl)-biotinamide. To a solution of 220 mg (0.467 mmol) of N,N-bis-(6-hexanoyl)- biotinamide in 3 ml of dry dimethylformamide was added 160 mg (1.39 mmol) of N-hydroxysuccinimide followed by 210 mg (1.02 mmol) of dicyclohexyl-carbodiimide. The mixture was stirred at 15-25°C for 17 hours and then concentrated. The residue was chromatographed on silica gel, eluting with 0.1:20:80 acetic acid/methanol/ethyl acetate, to afford 148 mg of the product as a foamy off-white solid (48%) : H-NMR (DMSO) 6.39 (1H, s) , 6.32 (1H, s) , 4,29 (1H, m) , 4,12 (1H, m) , 3.30-3.03 (5H, ) , 2.81 (9H, dd and s) , 2.67 (4H, m) , 2.57 (1H, d) , 2.25 (2H, t) , 1.75-1.20 (18H, Hi) ,* TLC-R,* 0.37 (0.1:20:80 acetic acid/methanol/ethyl acetate). 6. Preparation of N,N-bis-(6-hexanoylamidobenzyl- DOTA)-biotinamide. To a mixture of 15 mg of DOTA- benzylamine and 6.0 mg of N' ,N'-bis-(N-hydroxy- succinimidyl 6-hexanoyl)-biotinamide in 1.0 ml of dry dimethylformamide was added 0.5 ml of dry pyridine. The mixture was stirred at 45-50"C for 4.5 hours and at 15-25 * C for 12 hours. The mixture was concentrated and the residue chromatographed on a 2.1 x 2.5 cm octadecylsilyl (ODS) reverse-phase preparative HPLC column eluting with a —20 minute gradient profile of 0.1:95:5 to 0.1:40:60 trifluoroacetic acid:water:acetonitrile at 13 ml/minute to afford the desired product. The retention time was 15.97 minutes using the aforementioned gradient at a flow rate of 1.0 ml/minute on a 4.6 mm x 25 cm ODS analytical HPLC column. F. Synthesis of an N-methyl-glycine linked conjugate. A reaction scheme for this synthesis is shown below. The N-methyl glycine-linked DOTA-biotin conjugate was prepared by an analogous method to that used to prepare D-alanine-linked DOTA-biotin conjugates. N- methyl-glycine (trivial name sarcosine, available from Sigma Chemical Co.) was condensed with biotin-NHS ester in DMF and triethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotin was then activated with EDCI and NHS. The resultant NHS ester was not isolated and was condensed in situ with DOTA- aniline and excess pyridine. The reaction solution was heated at 60"C for 10 minutes and then evaporated. The residue was purified by preparative HPLC to give [ (N-methyl-N-biotinyl) -N-glycyl]-aminobenzyl-DOTA. 1. Preparation of (N-methyl)glycyl biotin. DMF (8.0 ml) and triethylamine (0.61 ml, 4.35 mmol) were added to solids N-methyl glycine (182 mg, 2.05 mmol) and N-hydroxy-succinimidyl biotin (500 mg, 1.46 mmol) . The mixture was heated for 1 hour in an oil bath at 85"C during which time the solids dissolved producing a clear and colorless solution. The solvents were then evaporated. The yellow oil residue was acidified with glacial acetic acid, evaporated and chromatographed on a 27 mm column packed with 50 g silica, eluring with 30% MeOH/ΞtOAc 1% HOAc to give the proάuc" as a white solid (3S3 mg) in 66% yield. H-NMR (DMSO) : 1.18-1.25 (m, 6H, (CH 2 ) 3 ) , 2.15, 2.35 (2 t's, 2H, CH : C0) , 2.75 ( , 2H, SCH 2 ) , 2.80, 3.00 (2 S's, 3H, NCH 3 ) , 3.05-3.15 ( , 1H, SCH) , 3.95, 4.05 (2 s's, 2H, CH 2 N) , 4.15, 4.32 (2 m'Ξ, 2H, 2CHN's) , 6.35 (s, NH) , 6.45 (s, NH) . 2. Preparation of [ (N-methyl-N-biotinyl)glycyl] aminobenzyl-DOTA. N-hydroxysuccinimide (10 mg, 0.08 mmol) and EDCI (15 mg, 6.08 mmol) were added to a solution of (N-methylglycyl biotin (24 mg, 0.08 mmol) in DMF (1.0 ml) . The solution was stirred at 23 'C for 64 hours. Pyridine (0.8 ml) and aminobenzyl-DOTA (20mg, 0.04 mmol) were added. The mixture was heated in an oil bath at 63 'C for 10 minutes, then stirred at 23°C for 4 hours. The solution was evaporated. The residue was purified by preparative HPLC to give the product as an off white solid (8 mg, 0.01 mmol) in 27% yield. H-NMR (D 2 0) : 1.30-1.80 (m, 6H) , 2.40, 2.55 (2 t's, 2H, CH,CO) , 2.70-4.2 (complex multiplet) , 4.35 (m, CHN) , 4.55 (m, CHN) , 7.30 (m, 2H, benzene hydrogens), 7.40 (m, 2H, benzene hydrogens) . G. Synthesis of a short chain amine-linked conjugate with a reduced biotin carboxy group. A two- part reaction scheme is shown below. (biotin - OTs) (:F p 3,-Ϊ !O The biotin carboxyl group is reduced with diborane in THF to give a primary alcohol. Tosylation of the alcohol with tosyl chloride in pyridine affords the primary tosylate. Aminobenzyl DOTA is acylated with trifluoroacetic anhydride in pyridine to give (N- trifluoroacetyl)aminobenzyl-DOTA. Deprotonation with 5.0 equivalents of sodium hydride followed by displacement of the biotin tosylate provides the (N- trifluoracetamido-N-descarboxylbiotinyl)aminobenzyl- DOTA. Acidic cleavage of the N-trifluoroacetamide group with HCl(g) in methanol provides the amine- linked DOTA-biotin conjugate. EXAMPLE XVI Clearing Agent Evaluation Experimentation The following experiments conducted on non-tumor- bearing mice were conducted using female BALB/c mice (20-25 g) . For tumor-bearing mice experimentation, female nude mice were injected subcutaneously with LS- 180 tumor cells, and, after 7 d, the mice displayed 50-100 mg tumor xenografts. The monoclonal antibody used in these experiments was NR-LU-10. When radiolabeled, the NR-LU-10-streptavidin conjugate was radiolabeled with 1-125 using procedures described herein. When radiolabeled, PIP-biocytin was labeled with 1-131 or 1-125 using procedures described herein. A. Utility of Asialooroso ucoid-Biotin (AO-Bt) in Reducing Circulating Radioactivity from a Subsequently Administered Radiolabeled Biotin Ligand. Mice bearing LS-180 colon tumor xenografts were injected with 200 micrograms NR-LU-10 antibody-streptavidin (MAb-StrAv) conjugate at time 0, which was allowed to prelocalize to tumor for 22 hours. At that time, 20 micrograms of AO-Bt was administered to one group of animals. Two hours later, 90 micrograms of a radioisotope-bearing, ligand-containing small molecule (PIP-biotin-dextran prepared as discussed in part B hereof) was administered to this group of mice and also to a group which had not received AO-Bt. The results of this experiment with respect to radiolabel uptake in tumor and clearance from the blood indicated that tumor- targeting of the radiolabeled biotin-containing conjugate was retained while blood clearance was enhanced, leading to an overall improvement in amount delivered to target/amount located in serum. The AUC tumor/AUC blood with clearing agent was 6.87, while AUC tumor/AUC blood without clearing agent was 4.45. Blood clearance of the circulating MAb-StrAv conjugate was enhanced with the use of clearing agent. The clearing agent was radiolabeled in a separate group of animals and found to bind directly to tumor at very low levels (1.7 pmol/g at a dose of 488 total pmoles (0.35%ID/g) , indicating that it does not significantly compromise the ability of tumor-bound MAb-StrAv to bind subsequently administered radiolabeled ligand. B. Preparation Protocol for PIP-Biotin-Dextran. A solution of 3.0 mg biotin-dextran, lysine fixable (BDLF, available from Sigma Chemical Co., St. Louis, Missouri, 70,000 dalton molecular weight with approximately 18 biotins/molecule) in 0.3 ml PBS and 0.15 ml 1 M sodium carbonate, pH 9.25, was added to a dried residue (1.87 mCi) of N-succinimidyl p-I-125- iodobenzoate prepared in accordance with Wilbur, et al., J. Nucl. Med.. 30: 216-226, 1989. C. Dosing Optimization of AO-Bt. Tumored mice receiving StrAv-MAb as above, were injected with increasing doses of AO-Bt (0 micrograms, 20 micrograms, 50 micrograms, 100 micrograms and 200 micrograms) . Tumor uptake of I-131-PIP-biocytin (5.7 micrograms, administered 2 hours after AO-Bt administration) was examined. Increasing doses of AO- Bt had no effect on tumor localization of MAb-StrAv. Data obtained 44 hours after AO-Bt administration showed the same lack of effect. This data indicates that AO-Bt dose not cross-link and internalize MAb- StrAv on the tumor surface, as had been noted for avidin administered following biotinylated antibody. PIP-biocytin tumor localization was inhibited at higher doses of AO-Bt. This effect is most likely due to reprocessing and distribution to tumor of biotin used to derivatize AO-Bt. Optimal tumor to blood ratios (% injected dose of radiolabeled ligand/gram weight of tumor divided by % injected dose of radioligand/gram weight of blood were achieved at the 50 microgram dose of AO-Bt. Biodistributions conducted following completion of the protocols employing a 50 microgram AO-Bt dose revealed low retention of radiolabel in all non-target tissues (1.2 pmol/g in blood; 3.5 pmol/gram in tail; 1.0 pmol/g in lung; 2.2 pmol/g in liver; 1.0 pmol/g is spleen; 7.0 pmol/g in stomach; 2.7 pmol/g in kidney; and 7.7 pmol/g in intestine). With 99.3 pmol/g in tumor, these results indicate effective decoupling of the PIP-biocytin biodistribution from that of the MAb- StrAv at all sites except tumor. This decoupling occurred at all clearing agent doses in excess of 50 micrograms as well. Decreases in tumor localization of PIP-biocytin was the significant result of administering clearing agent doses in excess of 50 micrograms. In addition, the amount of PIP-biocytin in non-target tissues 44 hours after administration was identical to localization resulting from administration of PIP-biocytin alone (except for tumor, where negligible accretion was seen when PIP- biocytin was administered alone) , indicating effective decoupling. D. Further Investigation of Optimal Clearing Agent Dose. Tumored mice injected with MAb-StrAv at time 0 as above; 50 micrograms of AO-Bt at time 22 hours; and 545 microcuries of I-131-PIP-biocytin at time 25 hours. Whole body radiation was measured and compared to that of animals that had not received clearing agent. 50 micrograms of AO-Bt was efficient in allowing the injected radioactivity to clear from the animals unimpeded by binding to circulating MAb- StrAv conjugate. Tumor uptake of I-131-PIP-biocytin was preserved at the 50 microgram clearing agent dose, with AUC tumor/AUC blood of 30:1 which is approximately 15-fold better than the AUC tumor/AUC blood achieved in conventional antibody-radioisotope therapy using this model. E. Galactose- and Biotin-Derivatization of Human Serum Albumin (HSA) . HSA was evaluated because it exhibits the advantages of being both inexpensive and non-immunogenic. HSA was derivatized with varying levels of biotin (1-about 9 biotins/molecule) via analogous chemistry to that previously described with respect to AO. More specifically, to a solution of HSA available from Sigma Chemical Co. (5-10 mg/ml in PBS) was added 10% v/v 0.5 M sodium borate buffer, pH 8.5, followed by dropwise addition of a DMSO solution of NHS-LC-biotin (Sigma Chemical Co.) to the stirred solution at the desired molar offering (relative molar equivalents of reactants) . The final percent DMSO in the reaction mixture should not exceed 5%. After stirring for 1 hour at room temperature, the reaction was complete. A 90% incorporation efficiency for biotin on HSA was generally observed. As a result, if 3 molar equivalences of the NHS ester of LC-biotin was introduced, about 2.7 biotins per HSA molecule were obtained. Unreacted biotin reagent was removed from the biotin-derivatized HSA using G-25 size exclusion chromatography. Alternatively, the crude material may be directly galactosylated. The same chemistry is applicable for biotinylating non-previously biotinylated dextran. HSA-biotin was then derivatized with from 12 to 15 galactoses/molecule. Galactose derivatization of the biotinylated HSA was performed according to the procedure of Lee, et al., Biochemistry, 15: 3956, 1976. More specifically, a 0.1 M methanolic solution of cyanomethyl-2,3,4,6-tetra-O-acetyl-l-thio-D- galactopyranoside was prepared and reacted with a 10% v/v 0.1 M NaOMe in methanol for 12 hours to generate the reactive galactosyl thioimidate. The galactosylation of biotinylated HSA began by initial evaporation of the anhydrous methanol from a 300 fold molar excess of reactive thioimidate. Biotinylated HSA in PBS, buffered with 10% v/v 0.5 M sodium borate, was added to the oily residue. After stirring at room temperature for 2 hours, the mixture was stored at 4°C for 12 hours. The galactosylated HSA-biotin was then purified by G-25 size exclusion chromatography or by buffer exchange to yield the desired product. The same chemistry is exploitable to galactosylating dextran. The incorporation efficiency of galactose on HSA is approximately 10%. 70 micrograms of Galactose-HSA-Biotin (G-HSA-B) , with 12-15 galactose residues and 9 biotins, was administered to mice which had been administered 200 micrograms of StrAv-MAb or 200 microliters of PBS 24 hours earlier. Results indicated that G-HSA-B is effective in removing StrAv-MAb from circulation. Also, the phar acokinetics of G-HSA-B is unperturbed and rapid in the presence or absence of circulating MAb-StrAv. F. Non-Protein Clearing Agent. A commercially available form of dextran, molecular weight of 70,000 daltons, pre-derivatized with approximately 18 biotins/molecule and having an equivalent number of free primary amines was studied. The primary amine moieties were derivatized with a galactosylating reagent, substantially in accordance with the procedure therefor described above in the discussion of HSA-based clearing agents, at a level of about 9 galactoses/molecule. The molar equivalence offering ratio of galactose to HSA was about 300:1, with about one-third of the galactose being converted to active form. 40 Micrograms of galactose-dextran-biotin (GAL- DEX-BT) was then injected i.v. into one group of mice which had received 200 micrograms MAb-StrAv conjugate intravenously 24 hours earlier, while 80 micrograms of GAL-DEX-BT was injected into other such mice. GAL- DEX-BT was rapid and efficient at clearing StrAv-MAb conjugate, removing over 66% of circulating conjugate in less than 4 hours after clearing agent administration. An equivalent effect was seen at both clearing agent doses, which correspond to 1.6 (40 micrograms) and 3.2 (80 micrograms) times the stoichiometric amount of circulating StrAv conjugate present. G. Dose Ranging for G-HSA-B Clearing Agent. Dose ranging studies followed the following basic format: 200 micrograms MAb-StrAv conjugate administered; 24 hours later, clearing agent administered; and 2 hours later, 5.7 micrograms PIP-biocytin administered. Dose ranging studies were performed with the G- HSA-B clearing agent, starting with a loading of 9 biotins per molecule and 12-15 galactose residues per molecule. Doses of 20, 40, 70 and 120 micrograms were administered 24 hours after a 200 microgram dose of MAb-StrAv conjugate. The clearing agent administrations were followed 2 hours later by administration of 5.7 micrograms of I-131-PIP- biocytin. Tumor uptake and blood retention of PIP- biocytin was examined 44 hours after administration thereof (46 hours after clearing agent administration) . The results showed that a nadir in blood retention of PIP-biocytin was achieved by all doses greater than or equal to 40 micrograms of G-HSA- B. A clear, dose-dependent decrease in tumor binding of PIP-biocytin at each increasing dose of G-HSA-B was present, however. Since no dose-dependent effect on the localization of MAb-StrAv conjugate at the tumor was observed, this data was interpreted as being indicative of relatively higher blocking of tumor- associated MAb-StrAv conjugate by the release of biotin from catabolized clearing agent. Similar results to those described earlier for the asialoorosomucoid clearing agent regarding plots of tumor/blood ratio were found with respect to G-HSA-B, in that an optimal balance between blood clearance and tumor retention occurred around the 40 microgram dose. Because of the relatively large molar amounts of biotin that could be released by this clearing agent at higher doses, studies were undertaken to evaluate the effect of lower levels of biotinylation on the effectiveness of the clearing agent. G-HSA-B, derivatized with either 9, 5 or 2 biotins/molecule, was able to clear MAb-StrAv conjugate from blood at equal protein doses of clearing agent. All levels of biotinylation yielded effective, rapid clearance of MAb-StrAv from blood. Comparison of these 9-, 5-, and 2-biotin- derivatized clearing agents with a single biotin G- HSA-B clearing agent was carried out in tumored mice, employing a 60 microgram dose of each clearing agent. This experiment showed each clearing agent to be substantially equally effective in blood clearance and tumor retention of MAb-StrAv conjugate 2 hours after clearing agent administration. The G-HSA-B with a single biotin was examined for the ability to reduce binding of a subsequently administered biotinylated small molecule (PIP-biocytin) in blood, while preserving tumor binding of PIP-biocytin to prelocalized MAb-StrAv conjugate. Measured at 44 hours following PIP-biocytin administration, tumor localization of both the MAb-StrAv conjugate and PIP- biocytin was well preserved over a broad dose range of G-HSA-B with one biotin/molecule (90 to 180 micrograms) . A progressive decrease in blood retention of PIP-biocytin was achieved by increasing doses of the single biotin G-HSA-B clearing agent, while tumor localization remained essentially constant, indicating that this clearing agent, with a lower level of biotinylation, is preferred. This preference arises because the single biotin G-HSA-B clearing agent is both effective at clearing MAb-StrAv over a broader range of doses (potentially eliminating the need for patient-to-patient titration of optimal dose) and appears to release less competing biotin into the systemic circulation than the same agent having a higher biotin loading level. Another way in which to decrease the effect of clearing agent-released biotin on active agent-biotin conjugate binding to prelocalized targeting moiety- streptavidin conjugate is to attach the protein or polymer or other primary clearing agent component to biotin using a retention linker. A retention linker has a chemical structure that is resistant to agents that cleave peptide bonds and, optionally, becomes protonated when localized to a catabolizing space, such as a lysosome. Preferred retention linkers of the present invention are short strings of D-amino acids or εmall molecules having both of the characteristics set forth above. An exemplary retention linker of the present invention is cyanuric chloride, which may be interposed between an epsilon amino group of a lysine of a proteinaceous primary clearing agent component and an amine moiety of a reduced and chemically altered biotin carboxy moiety (which has been discussed above) to form a compound of the structure set forth below. When the compound shown above is catabolized in a catabolizing space, the heterocyclic ring becomes protonated. The ring protonation prevents the catabolite from exiting the lysosome. In this manner, biotin catabolites containing the heterocyclic ring are restricted to the εite(s) of catabolism and, therefore, do not compete with active-agent-biotin conjugate for prelocalized targeting moiety- streptavidin target sites. Comparisons of tumor/blood localization of radiolabeled PIP-biocytin observed in the G-HSA-B dose ranging studies showed that optimal tumor to background targeting was achieved over a broad dose range (90 to 180 micrograms) , with the reεults providing the expectation that even larger clearing agent doses would also be effective. Another key result of the dose ranging experimentation is that G- HSA-B with an average of only 1 biotin per molecule is preεu ably only clearing the MAb-StrAv conjugate via the Ashwell receptor mechanism only, because too few biotins are present to cause cross-linking and aggregation of MAb-StrAv conjugates and clearing agents with such aggregates being cleared by the reticuloendothelial system. H. Tumor Targeting Evaluation Using G-HSA-B. The protocol for this experiment was as follows: Time 0: administer 400 micrograms MAb-StrAv conjugate; Time 24 hours: administer 240 micrograms of G- HSA-B with one biotin and 12-15 galactoses and Lu-177 is complexed with the DOTA chelate using Known techniques therefor. Efficient delivery of the Lu-177-DOTA-biotin small molecule was observed, 20-25 % injected dose/gram of tumor. These values are equivalent with the efficiency of the delivery of the MAb-StrAv conjugate. The AUC tumor/AUC blood obtained for thiε non- optimized clearing agent dose was 300% greater than that achievable by comparable direct MAb-radiolabel administration. In addition, the HSA-based clearing agent is expected to exhibit a low degree of immunogenicity in humans. Example XVII Synthesis of DOTA Chelates A. Peptide cyclization. N-Boc-triglycine. To a suspension of 18.92 g (100 mmole) of triglycine (available from Sigma Chemical Company) in 100 ml dioxane and 100 ml water was added 27.88 ml (200 mmole, 2.0 equivalents) of triethylamine and 27.1 g (110 mmole, 1.1 equivalents) of 2-[tert- butoxycarbonyloxyimino) -2-phenylacetonitrile (available from Fluka, New York) . The reaction mixture was stirred at room temperature overnight. Water (65 ml) was added to the mixture, and the resultant mixture was extracted with ether (4x65 ml) ant EtoAc (1x70 ml) . The aqueous layer was adjusted to pH 1.5 with 6M HCl at 0°C and then extracted with EtOAc (25 x 100 ml) by the salt-out technique. A substantial amount of product precipitated out of the EtOAc extraction and was collected by filtration. The EtOAc filtrate was reduced to 1/5 of its original volume under vacuum to produce additional precipitate. The precipitate was collected by filtration and the above procedure (reduction of volume to 1/5) was repeated. The precipitates were combined and dried under vacuum to give 23.82 g of product. (82.3% yield) ! H-NMR (CD 3 CN,£): 7.05 (bs, 2H, NH) , 5.70 (bε, IH, NH) , 3.82 (dd, 4H, CH 2 ) , 3.64 (d, 2H, CH 2 ) and 1.92 (ε, 9H, t-butyl) . N-Boc-triglycine N-hydroxyεuccinimide eεter. To a turbid εolution of 17.357 g (60 mmole) of N-BOC- triglycine and 13.5 g (65.4 mmole, 1.09 equivalentε) of 1,3-dicyclohexylcarbodiimide (DCC) in 300 ml anhydrouε dioxane waε added 6.906 g (60 mmole, 1 equivalent) of N-hydroxysuccinimide in one portion. The reaction mixture was stirred at room temperature overnight under nitrogen. DCU, dicyclohexylurea, was removed by filtration, and the filtrate was concentrated to dryness under vacuum. The resulting foamy product was triturated with hexane and then further dried under vacuum to give 27.68 g of crude product. (> 100% crude yield) -NMR εtudieε of thiε crude product εhowed that it might be formed in a 2:1 complex with dioxane. *H-NMR (CDC1 3 ,5): 7.34 and 7.06 (2m, NH) , 5.40 (t, NH) , 4.36 (d, CH 2 ) , 4.04 (d, CH 2 ) 3.82 (d, CH 2 ) , 3.71 (s, dioxane), 2.85 (s, CH 2 ) , and 1.70 (s, t-butyl) . N-Boc-triglycine-p-nitro-phenylalanine. To a εtirred solution of 30 mmole of N-boc-triglycine N- hydroxysuccinimide ester and 8.828 g (42 mmole, 1.4 equivalents) of p-nitrophenylalanine (available from Aldrich Chemical Co. , Milwaukee) in 56 ml of DMF and 114 ml water was added 5.04 g (60 mmole, 2.0 equivalents) of NaHC0 3 . The reaction mixture was εtirred at room temperature for 5 hours. Precipitate was filtered off, and the filtrate waε concentrated under vacuum. The resulting residue was disεolved in 250 ml of water and acidified with 6M HCl at 0°C to pH 1.4. The aqueous layer was extracted with EtOAc (6x150 ml) . The organic layers were combined and evaporated to dryness under vacuum. The crude product thus obtained was crystallized in 2-propanol to form the product. α H-NMR (CD 3 CN,S) 8.12 and 7.48 (2d, 4H, ArH), 7.20 (m, 3H, NH) , 5.78 (m, IH, NH) , 4.64 (m, IH, CH) , 3.72 (IB, 6H, CH 2 ) , 3.20 (m, 2H, CH 2 Ar) , and 1.93 (s, 9H, t-butyl) . The Boc protecting group of the linear tetrapeptide N-Boc-triglycine-p-nitro-phenylalanine is cleaved using 50% trifluoroacetic acid in CH 2 C1 2 at room temperature for 30 minutes to form triglycine-p- nitrophenyl-alanine. Peptide cyclization iε then achieved by mixing triglycine-p-nitrophenyl-alanine with 1.2 equivalents of diphenylphosphoroazide (DPPA) and 10 equivalents of sodium bicarbonate in anhydrous DMF under high dilution conditions (0.007M of triglycine-p-nitrophenyl-alanine) at 4°C for three days. Water and Bio-RAd AG501-X8 mixed bed ion exchange resin are added to the reaction mixture, and the mixture is stirred at room temperature for 3 hours. The reεin is separated by filtration, and the filtrate is concentrated in vacuo to give a crude product. The crude product is extracted with HOAc twice, and the HOAc extractions are combined and dried under vacuum to give the cyclo-tetrapeptide 2-(4- nitrobenzyl)-3,6,9,12-tetraoxo-l,4,7, 10-tetraaza- cyclododecane. Aε a conεequence of the relatively poor εolubility of the nitro-derivatized compound 2-(4-nitrobenzyl- 3,6,9, 12-tetraoxo-l,4,7, 10-tetraazacyclododecane, alternative intermediates may be employed in the syntheεiε of nitrobenzyl-DOTA. For example, the nitro group may be εubstituted by hydrogen or RNH- where R is an amino protecting group. Preferred R groups include tolueneεulfonyl, isonicotinyl-carbamate and 9- fluorenylmethyl carbamate. These alternative compounds can be prepared analogously to the nitro derivative by subεtituting p-nitrophenylalanine with phenylalanine (available from Sigma Chemical Co., St. Louiε Miεεouri for hydrogen substitution) or the protected p-aminophenylalanine (for RNH- substitution and prepared, for example, as follows: p-nitrophenyl¬ alanine iε reacted with BOC-ON in dioxane and water in the preεence of Et 3 N at room temperature overnight to afford N-Boc-p-nitrophenylalanine. The nitro group of N-Boc-p-nitro-phenylalanine iε reduced with 10% Pd-C under 40 pεi of H 2 in MeOH for 2 hours to produce the α-N-Boc-p-amino-phenylalanine. Subsequent protection of the para-amino group is achieved by treating α-N- Boc-p-amino-phenylalanine with 4-methoxy-2,3,6- trimethylbenzene-εulfonylchloride (MtrCl) and Et 3 N in CH 2 C1 2 at room temperature for 17 hourε. The Boc protecting group of the alpha-amino group of α-N-Boc- p-Mtr-amidophenyl-alanine is then cleaved by 50% TFA in CH 2 C1 2 at room temperature for 30 minutes to give the Mtr protected p-aminophenylalanine. ) When hydrogen subεtitution iε employed, the nitro group is introduced later in the syntheεiε (e.g. , following cyclization and reduction to form the tetraamine product) . When the protected amino intermediate iε employed, the amine protecting group iε inert to diborane reduction. Conεequently, alkylation with bromoacetic acid followed by deprotection of the para-amino protecting group afford an anilino-DOTA product. The tetraamine 2-(p-nitrophenyl) -1,4,7, 10- tetraaza-cyclododecane iε formed by reducing 2-(4- nitrobenzyl) -3,6,9, 12-tetraoxo-l,4,7, 10- tetraazacyclododecane with 12 equivalentε of diborane in THF at 50°C for two dayε. Hydrolyεiε of the reεulting borane complex iε accompliεhed in MeOH εaturated with HCl gaε. 2-(p-nitrophenyl) -1,4,7,10- tetraazacyclododecane is treated with 4.4 equivalents of bromoacetic acid in water at pH 10 at 70°C for one day to give crude product. The product p-nitrobenzyl- DOTA is obtained after anion-exchange column chromatography with elution of increasing concentration of ammonium acetate, for example. B. Diethylenetriamine and N-alkylated phenyl alanine route. N, N', N' ' '-triε-(p-tolueneεulfonyl) -diethylene triamine. A solution of IN sodium hydroxide is added to a solution of diethylene triamine (available from Aldrich Chemical Co., Milwaukee, WI) in water until the pH is 10. Then p-toluenesulfonyl chloride (1.4 mole equivalents) is added as a solid all at once. Additional IN sodium hydroxide is added as needed to maintain the pH of the reaction mixture between 10 and 12. When the pH ceases to change the mixture is filtered. The solid is rinsed with dichloromethane. The reεultant solid is dried in vacuo to give the product which, if necessary, may be purified by recrystallization. N-(p-toluenesulfonyl) -(4-nitrophenyl) -alanine. To a solution of 4-nitro-L-phenyl-alanine (available from Aldrich) (100g) in water (3.0 ml) and 0.95N NaOH (5.0 ml) was added p-tolueneεulfonyl chloride (1.27g) all at once. The reaction waε stirred at room temperature, and the pH was continuouεly monitored with a pH meter. After 2 hourε, the pH of the reaction had dropped from 10.5 to 8.3; additional 0.95N NaOH waε added to raiεe the pH to 10.4. After 2 more hourε, the pH waε 8.5, and additional 0.95N NaOH waε added to raiεe the pH to 11.0. The reaction mixture waε εtirred an additional 16 hourε at room temperature. The reaction mixture which contained a large amount of white solid, was diluted with water (30 ml) and CH 2 C1 2 (100 ml) and filtered. The solid was rinsed with CH 2 C1 2 . The filtrate was transferred to a separatory funnel. The CH 2 C1 2 layer, which contained p-tolueneεulfonyl chloride, waε εeparated and discarded. The aqueous and filtered εolid phaεes were combined, acidified with 1.0M HCl to pH 2, and filtered. The solid waε dried in vacuo to give 1.55 g product (98% yield) . 'H-NMR (CDC1 3 ) : 2.40 (s, 3H, CH 3 ) , 3.18 (dd, 2H, CH 2 ) , 4.25 (m, IH, CH) , 5.25 (d, IH, NH) , 7.25 (dd, 4H) , 7.60 (d, 2H) , and 8.05 (d, 2H) . N-(p-toluenesulfonyl) -N-(2-hydroxyethyl) -(4- nitrophenyl) -L-alanine. DMF was added to a mixture of N-tosyl-p-nitrophenylalanine (332 mg, 1.0 mmol), ethylene carbonate (440 mg, 5.0 mmol), and potasεium carbonate (690 mg, 5.0 mmol) and the resultant mixture was heated in an oil bath at 60°C for 19 hourε. The DMF waε evaporated under reduced preεεure. The reεidue waε diluted with 1.0M HCl (30 ml) and extracted with methylene chloride (3 x 30 ml) . The combined CH 2 C1 2 extracts were dried (MgS0 4 ) and evaporated to give 430 mg brown oil which waε purified by flaεh chromatography on εilica gel with 99:1 ethyl acetate:acetic acid to give the product in 40% yield. l E NMR (CDC1 3 ) : 2.40 (s, 3H, CH 3 ) , 3.20 (dd, 2H, ArCH 3 ) , 4.32 (m, IH, CHN), 4.50-4.85 (m, 3H) , 5.70 (m, IH) , 7.17 (d, 2H) , 7.30 (d, 2H) , 7.55 (d, 2H) , 8.03 (d, 2H) . IR (CDC1 3 ) : 3300, 1755, 1670, 1420, 1160 cm' l-(4-Nitrobenzyl) -N-tosyl-iminodiethanol. 1.0M Diborane in THF is added to a solution of N-(p- tolueneεulfonyl) -N-(2-hydroxyethyl) -(4-nitrophenyl) -L- alanine in THF. The reaction iε stirred at 23°C for 4 hours. Methanol is added and the solution is evaporated. The residue iε treated with methanol and evaporated twice more to dryneεs. The product is purified by chromatography on silica gel (Silica gel 60, EM Science, Gibbstown, NJ) . N-[2-(p-tolylεulfonyl) oxy]ethyl-N-[2-p- tolylεulfonyl)oxy]-1-(4-nitrobenzyl)ethyl]-p- tolueneεulfonamide. P-toluenesulfonyl chloride is added to a solution of l-(4-nitrobenzyl) -N-(p- tolueneεulfonyl)-iminodiethanol in dichloromethane and triethylamine. The solution is stirred at 23°C for 20 hours, diluted with methylene chloride and washed twice with 0.1M HCl. The methylene chloride extracts are dried (MgS0 4 ) and evaporated. The residue is purified by chromatography on εilica gel (Silica gel 60, EM Science) . (S)-2-(p-nitrobenzyl)-N,N ,N' ' ,N' ' '-tetrakiε- (tolylsulfonyl)-1,4,7,10-tetraazacyclododecane. Equimolar amounts of N-[2-(p-tolylsulfonyl)oxy]ethyl- N-[2-(p-tolylεulfonyl)oxy]-l-(4-nitrobenzyl)ethyl]-p- toluenesulfonamide and N,N' ,N' ' '-tris-(p-toluene sulfonyl)-diethylenetriamine are dissolved in DMF. Two equivalents of cesium carbonate are added and the reaction is heated at 75°C for 14 hours. The solvent is removed under reduced pressure, and the residue iε dissolved in CHC1 3 and washed with 0.1M HCl three times. The CHC1 3 phase is dried (MgS0 4 ) and evaporated. The residue is purified on silica gel (Silica gel 60, EM Science) with CHC1 3 . Conversion of this product to nitro-benzyl-DOTA is accomplished via the procedure discusεed in Renn et al., Biocon . Chem. , 3.:563-569, 1992 or as discussed in subpart C εet forth below. In prior art εynthetic routes, N-tosylation of an aminoalcohol proved to be problematic and proceeded in low yield. The synthetic route set forth above, in contrast, features selective N-toεylation prior to introduction of the alcohol. The subsequent O- toεylation εtep iε leεs εusceptible to potential side reactionε ariεing from nucleophilic attack of the amino group on the O-tosylate. Also, this synthetic route has the advantage that the electron withdrawing nitrobenzyl group is substituted on the electrophile (i.e. , the leaving group half of the molecule undergoing cyclization) , thus not impairing the nucleophilicity of the nucleophilic tosylamide half. When this substitution is reversed, the yield of the cyclization step is greatly reduced. This synthetic route includes seven steps and is, therefore, relatively efficient. C. Tosylation cyclization. N-Boc-triglycine-p-nitro-phenylalanine is prepared in accordance with the procedure described in subpart A of this Example. N-Boc-triglycine-p-nitro- phenylalanine iε firεt treated with HCl gaε-εaturated dioxane to cleave the Boc protecting group, forming a deprotected triamide intermediate. Thiε intermediate is then reduced with 13 equivalents of diborane in THF at refluxing temperature. Hydrolysiε of the reεultant borane complex with MeOH εaturated with HCl gas affords a hydroxy tetramine compound, 2-(p- nitrobenzyl)-3,6,9, 12-tetraazadodecanol. Toεylation of 2-(p-nitrobenzyl) -3, 6,9, 12-tetraaza- dodecanol iε carried out in a stepwise faεhion as set forth below. The hydroxy group is first transiently protected with a trimethylsilyl group by treating 2- (p-nitrobenzyl)- 3, 6, 9, 12-tetraazadodecanol with 1.2 equivalents of trimethylchlorosilane (available from Aldrich, Milwaukee, WI) and 2 equivalentε of triethylamine in THF at room temperature for 4 hourε. Solvent is removed from the resultant mixture under reduced presεure (e.g. , 1 torr) . The tranεient protected intermediate is reacted with 4 equivalents of p-toluenesulfonyl chloride and 4 equivalents of triethylamine in CH 2 C1 2 /CH 3 CN (2/1 v/v) at room temperature for 5 hours to afford an N-tosylated intermediate. The transient trimethylsilyl protecting group is then cleaved, and the free hydroxy derivative is converted to the O-toεylated, N-tosylated compound IX, 2-(p-nitrobenzyl) -N,N' ,N' ' ,N' ' ' ,0-pentakis(tolyl- sulfonyl) -3, 6,9,12-tetraazadodecanol, by reacting the N-tosylated intermediate with 1.1 equivalents of p- toluenesulfonic chloride and 1 equivalent of tri¬ ethylamine in CH 2 C1 2 . The transient protection of the hydroxy group is conducted to increase the yield of 2- (p-nitrobenzyl) -N,N' ,N",N'" ,θ-pentakis(tolyl- εulfonyl) -3,6,9,12-tetraazadodecanol by minimizing the undesired εide reactionε potentially arising from inter- and intra-molecular condensationε of partially toεylated intermediateε. The intramolecular cyclization step undertaken to give 2-(p-nitrobenzyl) -N,N' ,N' ' ,N" '-tetrakis (tolylsulfonyl) -1,4,7, 10-tetraazacyclododecane iε performed by treating 2-(p-nitrobenzyl)-N,N' ,N' ' , N' ' ' ,O-pentakis(tolylsulfonyl) -3,6,9,12-tetraaza- dodecanol with 1 equivalent of CsC0 3 in DMF under high dilution conditions (0.01M of 2-(p-nitrobenzyl) - N,N',N" , N'" ,0-pentakis(tolylsulfonyl) -3, 6,9, 12- tetraaza-dodecanol) at 60°C for 5 hours under nitrogen. The toεyl protecting groups of 2-(p- nitrobenzyl) -N,N' ,N' ' ,N' "-tetrakis (tolylsulfonyl) - 1,4,7,10-tetraazacyclododecane are removed using concentrated H 2 S0 4 at 100°C for 56 hours, affording 2- (p-nitrophenyl) -1,4,7, 10-tetraaza-cyclododecane. 2- (p-nitrophenyl) -1,4,7, 10-tetraaza-cyclododecane is treated with 4.4 equivalents of bromoacetic acid in water at pH 10 at 70°C for one day to give crude product. The product p-nitrobenzyl-DOTA is obtained after anion-exchange column chromatography with elution of increasing concentration of ammonium acetate, for example. D. Intramolecular cyclization of nitro-phenyl- alanine-(glycine),. (S) -p-nitrophenylalanylglycyl- glycylglycine is prepared in accordance with the method described in Renn and Meares, Biocon . Chem.. 3 . : 563-569, 1992. 2-(4-Nitrobenzyl)-3,6,9, 12-tetraoxo-l,4,7, 10- tetraazacyclododecane. Diphenylphosphorylazide (available from Aldrich, Milwaukee, WI) iε added to a εtirred solution of (S) -p-nitrophenyl-alanylglycyl- glycylglycine in DMF at 0°C. Triethylamine is then added to the reaction. Stirring at 0°C is continued for 4 hourε and is followed by stirring at room temperature for 14 hours. The solvent is removed under reduced presεure. The reεidue iε taken up in glacial acetic acid. The εlurry iε heated to 45°C and filtered. The εolid product iε collected by filtration. (S) -2-(p-nitrobenzyl) -1,4,7, 10-tetraaza- cyclododecane. Borane-pyridine (available from Aldrich) iε added to a εuεpension of 2-(4- nitrobenzyl) -3,6,9, 12-tetraoxo-l,4,7, 10- tetraazacyclododecane in pyridine. The mixture is heated under reflux at 120°C for 14 hours. The solvent is removed under reduced pressure. The residue is taken up in methanol and heated under reflux for 2 hours. The solvent is removed under reduced pressure. The residue is dissolved in methanol and re-evaporated. The solid reεidue iε purified by chromatography on reverεe phaεe Baker G18 silica gel available from J.T. Baker, Inc., Phillipsburg, NJ. Conversion of this product to nitro-benzyl-DOTA iε accomplished via the procedure discussed in Renn et al., Biocon . Chem.. 3 . :563-569, 1992 or as set forth is subpart A above. This synthetic route includes five stepε and is, therefore, quite efficient. In prior art synthetic routes, the yield-limiting step is an intermolecular cyclization, while the route described above involves intra-molecular cyclization. Generally, intra- molecular cyclizations proceed in higher yield than inter-molecular cyclizations. In addition, intra- molecular cyclizations do not require the use of high dilution techniques. E. Phenyl-alanine route. As an alternative to employing 4-nitrophenylalanine in synthetic routes to benzyl-substituted tetraazadodecane derivatives, phenylalanine may be used. Two advantageε may be gained by introducing the nitro group following cyclization. Firεt, the εolubility of the εynthetic intermediates in organic solvents may be improved. Second, cyclization step yield may be enhanced. See, for example, McMurry et al., Biocon . Chem.. 3_*. 108- 117, 1992. An exemplary syntheεiε of thiε type iε εet forth below, wherein phenylalanine (available from Aldrich) was substituted for nitrophenylalanine in Route A set forth above. Briefly, N-Boc-triglycyl-N- hydroxyεuccinimidyl eεter obtained aε set forth in εubpart A of thiε Example iε condenεed with phenylalanine to give N-Boc-triglycylphenyl- phenylalanine. The Boc protecting group iε cleaved with trifluoroacetic acid to give N-Boc-triglycine- phenylalanine. Intramolecular cyclization of 3 affords the benzyl-cyclododecane derivative 2- (benzyl) -3,6,9, 12-tetraoxo-l,4,7, 10- tetraazacyclododecane, which is nitrated with nitric acid in sulfuric acid to give 2-(4-nitrobenzyl)- 3,6,9,12-tetraoxo-l,4,7,10-tetraazacyclododecane. Alternatively, a milder nitration reagent, εuch as N0 2 + CF 3 S0 3 ", may be employed. The amide carbonyls are reduced with borane-pyridine at elevated temperature. Conversion of the tetraazacyclododecane to nitro¬ benzyl-DOTA is accomplished via the procedure diεcuεεed in Renn et al., Bioconj . Chem.. 3_:563-569, 1992 or aε discussed in subsection A set forth above. F. Phenyl-lactic acid route. Phenyl-lactic acid (available from Aldrich Chemical Co., Milwaukee, WI) is nitrated with nitric acid in sulfuric acid. P-nitrophenyl lactic acid iε eεterified with methanol and gaseous hydrochloric acid. Methyl-p-nitrophenyl lactate is condensed with ethylene diamine to provide the amide adduct, 3-(4- nitrophenyl)-2-hydroxy-N-(2-aminoethyl)propionamide. The free amino group of the amide adduct iε acylated with N-hydroxy succinimidyl-N-trifluoroacetyl glycylglycine, formed through the derivatization of diglycine (available from Sigma Chemical Co., St. Louis, Miεsouri) as deεcribed below. The amino group of diglycine iε protected with a BOC protecting group uεing BOC-ON (available from Aldrich Chemical Co. , Milwaukee, Wiεconsin) in aqueous DMF. The Boc- diglycine adduct is then converted to the N- hydroxyεuccinimidyl eεter employing dicyclohexylcarbodiimide and N-hydroxysuccinimide. The aforementioned free amino acylation affords 3- (4-nitrophenyl) -2-hydroxy-N-(trifluoroacetylglycyl- glycyl-2-aminoethyl)propionamide. The hydroxyl group of 3-(4-nitrophenyl) -2-hydroxy-N-(trifluoroacetyl- glycylglycyl-2-aminoethyl)propionamide is tosylated with TsCl in pyridine to give 3-(4-nitrophenyl) -2-(p- toluenesulfonyl)-oxy-N-(trifluoroacetylglycylglycyl-2- aminoethyl) -propionamide. The trifluoroacetylamide group of 3-(5-nitrophenyl) -2-(p-toluenesulfonyl) -oxy- N-(trifluoroacetylglycylglycyl-2-aminoethyl) - propionamide is deprotonated with sodium hydride and cyclized to give 2-(4-nitrobenzyl)-3,8,11-trioxo-N- trifluoroacetyl-1,4,7,10-tetraazadodecane. The trifluoroacetyl group of 2-(4-nitrobenzyl) -3,8,11- trioxo-N-trifluoroacetyl-1,4,7, 10-tetraaza- cyclododecane is cleaved with sodium hydroxide to afford a triamide product, 2-(4-nitrobenzyl)-3,8, 11- trioxo-l,4,7,10-tetraazacyclododecane. The triamide is reduced with borane to give 2-(p-nitrophenyl) - 1,4,7, 10-tetraazacyclododecane. Converεion of this product to nitro-benzyl-DOTA iε accomplished via the procedure discussed in the Renn et al. article referenced above or as discuεεed in εubsections A and C set forth above. This synthetic route employs a phenyl-lactic acid starting material and peptide synthesis steps that are synthetically facile. The intramolecular cyclization is not conducted under high dilution conditions and a cyclomonomer product is favored. EXAMPLE XVIII Y-90 Chelation and Radiolabeled Biotinylated Molecules A. Radiolyεiε Experimentation. The stability of Y-90-DOTA-biotin conjugate to radiolyεis waε examined at different levels of specific activity (from 10 mCi/mg to 875 mCi/mg) . Specific activity is the ratio of yttrium activity (mCi) to the mass of the DOTA-biotin conjugate (mg) . The effect of radioprotectants, such as gentisic acid and ascorbic acid was also examined at the same range of specific activity. The reεultε εummarized in Tables 5 and 6 were obtained by the procedure deεcribed below. To the deεired starting Y-90 activity, an appropriate amount of DOTA-biotin conjugate, disεolved in 0.5M, pH 5 ammonium acetate, waε added. If employed, a radioprotectant was either added to this mixture or added to the radiolabeled preparation following the radiolabeling procedure. The compoεition of each reaction mixture (e.g. , activity, chelate linker, total volume, radioprotectant and the like) were aε set forth in the Tables. A 2 ml centrifuge tube was used as a reaction vessel, and the reaction mixture was incubated at 80°C in a water bath for 30 minutes. Radiopurities were asεeεsed by HPLC at different time intervals. High-performance liquid chromatography (HPLC) was conducted with a Beckman Model HOB solvent delivery system and Beckman Model 170 radioisotpe detector. Reverεe-phaεe HPLC chromatography waε carried out uεing a Beckman ultraεphere (4.6 mm x 12.5 cm) C-18 column uεing a gradient εolvent εyεtem at a flow rate of 1.0 ml/min. Solvent A in the gradient was a 10 mM diethylenetriaminepenta-acetic acid calcium trisodium salt water solution. Solvent B was 60% acetonitrile in a 10 mM diethylenetriaminepenta-acetic acid calcium trisodium salt water solution. The gradient was increased to 75% B over 15 minutes and maintained at 75% B for an additional 15 minutes. The gradient waε decreased to the original 100% A over a 10 minute time period. Table 5 indicates resultε of experimentation when the radioprotectant iε added following radiolabeling, while Table 6 εhowε experimental reεults when the radioprotectant is added as a radiolabeling ingredient. Tables 5 and 6 demonεtrate the inεtability of Y-90-DOTA-biotin conjugate (with N-Me ligand indicating a conjugate wherein biotin iε bound to DOTA via an N-methyl-glycine linker as set forth in Example XV(F) above and LC ligand indicating a conjugate wherein biotin is bound to DOTA via -NH- (CH 2 ) 5 , long chain DOTA-biotin) to radiolysis as well as the stability of such conjugates provided by the radioprotectants aεcorbic acid and gentiεic acid. Low εpecific activity preparationε did not exhibit problematic radiolyεiε. High εpecific activity preparationε; however, exhibited radiolyεiε. Addition of either gentiεic acid or aεcorbic acid decreaεed radiolysis in high specific activity preparations, with ascorbic acid giving somewhat better results than gentisic acid. The radioprotectants, e.g.. gentisic acid or ascorbic acid, were added either upon completion of the radiolabeling procedure or as part of the radiolabeling reaction mixture. The preferred option was to employ the radioprotectant as part of the radiolabeling reaction mixture. B. Y-90 Chelation Procedure. Carrier free ^C^ (20-200 μL in 0.5N HCl) was obtained from DuPont (Wilmington, Delaware) . The carrier free molecule was diluted with ammonium acetate buffer (0.5M, pH 5) to a total volume of 0.4 mL. Fifty microliters (500 mg/mL) of aεcorbic acid and 50-100 μL (10 mg/mL) of DOTA- biotin conjugate prepared in accordance with Example XV(F) above are added to the buffered ^YCl j εolution. Aεcorbic acid is present aε an antioxidant to prevent radiolyεiε of the preparation aε discussed above. The mixture was incubated for one hour at 80°C. Upon completion of the incubation, 55 μL of 100 mM DTPA (diethylenetriamine-pentaacetic acid) is added to the mixture to chelate any unbound Y-90. The final preparation was diluted to 10 mL with 0.9% NaCl. C. Radiolysiε product and oxidation. N-methyl- glycine-DOTA biotin waε examined via HPLC for radiolyεiε in accordance with the following procedure. To a solution of 80 μg (0.1 μmol) of Y-90-labeled N- methyl-glycine-DOTA-biotin conjugate in 0.4 ml of 0.5M ammonium acetate, pH 5, was added 43 μl of 23 mM (1.0 μmol) aqueouε εodium periodate. The mixture was maintained at room temperature for 30 minutes prior to evaluation by HPLC as set forth above. The oxidized biotin-containing conjugate exhibited the same HPLC retention time as the radiolysiε product of the correεponding biotin-containing conjugate. The oxidized biotin-containing conjugate retained the ability to bind avidin. Thiε reεult waε further confirmed by Y-90-radiolabeled N-methyl-glycine-DOTA- εulfoxide biotin, which waε prepared by reaction of N- methyl-glycine-DOTA-biotin with εodium periodate aε set forth below. Consequently, the "radiolyεiε product" of the biotin-containing conjugate may represent an oxidized biotin-containing conjugate rather than a conjugate wherein Y-90 is eεcaping itε DOTA chelate. Accordingly, the oxidized biotin- containing conjugate may be employed in the practice of the preεent invention. Radiolysis may result in total decomposition of the product conjugate, rather than merely oxidation as set forth above, however. In such circumstanceε, the use of radioprotectants will be beneficial. Sulfoxobiotinyl-N-methyl-glycyl-aminobenzyl-DOTA. Sodium periodate (26 mg) waε added to a solution of biotinyl-N-methylglycyl-aminobenzyl-DOTA (10 mg) in water (1.0 ml) . After the reaction was maintained at 23°C for 30 minutes, NMR showed no remaining starting material as evidenced by downfield εhift of imide methine from 4.4 and 4.6 ppm (starting material) to 4.7 and 4.9 ppm (product) as well as by downfield shift of S-methylene from 2.4 and 2.6 ppm (starting material) to 2.5 and 2.7 ppm (product). The product waε purified by preparative HPLC. Analysis of the purified product on analytical HPLC showed a single peak (7.69 minutes) having a 2 minute shorter retention time than the non-oxidized starting material. Gradient 95% H 2 0.1TFA, 5% CH 3 CN to 50% H 2 0.1TFA, 50%CH 3 CN over 15 minutes. Kits containing one or more of the components described above are also contemplated. For instance, radiohalogenated biotin may be provided in a sterile container for use in pretargeting procedures. A chelate-biotin conjugate provided in a sterile container is suitable for radio etallation by the consumer; such kits would be particularly amenable for use in pretargeting protocolε. Alternatively, radiohalogenated biotin and a chelate-biotin conjugate may be vialed in a non-sterile condition for uεe aε a reεearch reagent. From the foregoing, it will be appreciated that, although εpecific embodimentε of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method of producing nitro-benzyl-DOTA via intramolecular tosylate cyclization comprising: transient protection of a hydroxy group; tosylation of amines; deprotection of the transiently protected hydroxy group; toεylation of the deprotected hydroxy group; and an intramolecular tosylate cyclization reaction to form a twelve-membered DOTA ring. 2. The method of claim 1 wherein the toεylate cyclization iε performed uεing 2-(p-nitrobenzyl) -N, N' ,N" ,N" ' ,0-pentakiε(tosyl-sulfonyl)-3,6,9,12- tetraazadodecanol to form a twelve-membered DOTA ring. 3. The method of claim 1 wherein the toεylate cyclization is performed using 11-(p-nitrobenzyl) - N,N',N",N'" ,0-pentakis(tosyl-sulfonyl)-3,6,9,12- tetraazadodecanol to form a twelve-membered DOTA ring. 4. A method of producing nitro-benzyl-DOTA via intramolecular toεylate cyclization comprising: selective tosylation of free amines; tosylation of a free hydroxyl group; and an intramolecular tosylate cyclization reaction to form a twelve-membered DOTA ring. 5. The method of claim 4 wherein the toεylation of the free amines iε conducted in the absence of tertiary amineε or aromatic amine baεeε. 6. The method of claim 5 wherein the toεylation of the hydroxyl group is achieved at a basic pH. 7. The method of claim 6 wherein the tosylation is performed in the presence of triethylamine. 8. A method of producing nitro-benzyl-DOTA via direct peptide cyclization comprising; treating an acyclic tetrapeptide which has an N- terminal amino group and a C-terminal carboxyl group with an activating agent εuch that said carboxyl group of said acyclic tetrapeptide becomes suεceptible to closure by εaid amino group; and allowing said acyclic tetrapeptide to cyclize to form a cyclic tetraamide. 9. The method of claim 8, wherein εaid activating agent iε benzotriazolyloxytris(dimethylamino)phoεphonium hexafluorophoεphate. 10. The method of claim 8, wherein said cyclization step is conducted at a temperature greater than 37°C. 11. The method of claim 8, wherein said acyclic tetrapeptide is p- nitrophenylalanylglycylglycylglycine. 12. The method of claim 8, wherein said cyclic tetraamide is 2-(p-nitrobenzyl) -3, 6, 9, 12-tetraoxo- 1, 4, 7, 10-tetraazacylcododecane. 13. A method of producing nitro-benzyl-DOTA via direct peptide cyclization comprising: treating an acyclic tetrapeptide which has an N- terminal glycyl amino group and a C-terminal p- nitrophenylalanyl carboxyl group with an activating agent such that said carboxyl group of said acyclic tetrapeptide becomes susceptible to closure by said amino group; and allowing said acyclic tetrapeptide to cyclize to form a cyclic tetraamide. 14. The method of claim 13, wherein said activating agent is benzotriazolyloxytriε(dimethylamino) hosphonium hexafluorophoεphate. 15. The method of claim 13, wherein said cyclization εtep is conducted at a temperature greater than 37°C. 16. The method of claim 13, wherein said acyclic tetrapeptide is glycylglycylglycyl-p- nitrophenylalanine. 17. The method of claim 13, wherein said cyclic tetraamide is 2-(p-nitrobenzyl) -3, 6, 9, 12-tetraoxo- 1, 4, 7, 10-tetraazacylcododecane. 18. A method of producing nitro-benzyl-DOTA via the select N-alkylation of a compound having a trifluoroacetamide group with an iodoacetyl derivative in the presence of base comprising: treating an acyclic tripeptide having an N- terminal amino group and a C-terminal carboxyl group with an activating agent such that said carboxyl group of εaid acyclic tetrapeptide becomes susceptible to closure by said amino group; and allowing said acyclic tripeptide to cyclize to form a cyclic tripeptide. 19. The method of claim 18, wherein said trifluoroacetamide group is N-BOC-glycyl- trifluoroacetylethylenediamine. 20. The method of claim 18, wherein said iodoacetyl derivative is iodoacetyl-p- nitrophenylalanine t-butyl ester. 21. The method of claim 18, wherein said acyclic tetrapeptide is glycyl-N-ethylenediamino-N'- trifluoroacetyl-N'-methylcarbonyl-p- nitrophenylalanine. 22. The method of claim 18, wherein said cyclic tripeptide is 2-(p-nitrobenzyl)-3, 6, 9, 12-trioxy-10- -trifluoroacetyl-1, 4, 7, 10-tetraazacyclododecane.

Inventors: GUSTAVSON LINDA M, THEODORE LOUIS J, YAU ERIC K, GUSTAVSON, LINDA, M., THEODORE, LOUIS, J., YAU, ERIC, K.
Applicants: NEORX CORP, NEORX CORPORATION
Application year: 1994
Main classification: C07K1
IPC codes: A61K49, C07K1, C07K7